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Diets high in fat seem to correspond with an increased risk of certain forms of cancer, including bladder BlCa. This preliminary study examined the expression and enzyme activity profile of the polyunsaturated fatty acid metabolizing enzyme 15-Lipoxygenase-1 (15-LO-1) in human tissues from normal bladder and bladder tumors (stages CIS-T3/T4). Human tissue samples from normal (donor) bladder and bladder tumors (stages CIS-T3/T4; non-Bacillus Calmette-Guerin-treated) were grossly microdissected and analyzed for 15-LO-1 protein expression [immunohistochemistry (IHC)/Western blot], mRNA expression (quantitative real-time polymerase chain reaction) and enzyme activity profiles. Our results demonstrated that 15-LO-1 expression (protein/mRNA) and enzyme activity varied with BlCa progression. Specifically, IHC analyses of 15-LO-1 protein levels revealed decreased expression with increased bladder tumor stage. In particular, a statistically significant decrease in 15-LO-1 expression in stage T3/T4 bladder tumors compared with normal tissues (P<0.001) was observed. In agreement with IHC results, Western blot, quantitative real-time polymerase chain reaction, and enzymatic activity analyses demonstrated increased 15-LO-1 protein, mRNA, and enzyme activity, respectively, in normal human bladder tissues in comparison with stage T3/T4 human bladder tumors. Our finding of variable 15-LO-1 expression and enzyme activity in bladder tissues suggests a role for 15-LO-1 in bladder carcinogenesis.
(C) 2008 Lippincott Williams & Wilkins, Inc.
B7-H4, a member of the B7 family, is involved in the regulation of antigen-specific immune responses. Its expression in a range of breast pathology and correlation to the number of CD3+ and CD8+ tumor infiltrating T-lymphocytes in invasive carcinomas were explored. The proportion of B7-H4 positive cells, staining pattern, and intensity were evaluated within diagnostic groups (normal and benign lesional, potentially premalignant and in situ carcinoma, and invasive carcinoma) on archival tissue blocks by immunohistochemistry. The proportion and intensity of B7-H4 expression was progressively increased across the major diagnostic groups. There was a significant association between a high proportion of B7-H4 positive cells in invasive ductal carcinomas and decreased number of tumor infiltrating lymphocytes (P=0.002). The cellular distribution of B7-H4 appears altered in the spectrum of normal to malignant breast. Its overexpression may help cancers avoid immune detection.
(C) 2007 Lippincott Williams & Wilkins, Inc.
Background: The aim of this study was to investigate prognostic factors influencing the survival of synovial sarcoma, including the debated role of SYT-SSX fusion type and the newly suggested immunohistochemical marker ezrin.
Patients and Methods: From 1984 to 2005, 45 patients-25 men (56%) and 20 women (44%) with a median age of 31 (range: 2 to 81) years-were diagnosed with a synovial sarcoma. Age at diagnosis, tumor site, tumor size, tumor histology (biphasic vs. monophasic), mitotic count, necrosis, histologic grade, SYT-SSX fusion type, and ezrin immunostaining were analyzed for influence on survival by univariate and multivariate methods.
Results: The median follow-up for all patients was 55 (range: 2 to 238) months. Five patients had metastatic disease at the time of presentation. Five-year disease-specific survivals (DSS) were 67% overall and 72% for the 40 patients presenting with localized disease at diagnosis. Nineteen patients (48%) developed metastases during follow-up. Five-year metastasis-free survival (MFS) for the 40 patients with localized disease at diagnosis was 60% and the 10-year MFS was 52%. Disease stage at presentation, tumor size >5 cm, and histologic grade 3 were univariate significant factors associated with a worse DSS. Age >=30 years, tumor size >5 cm, necrosis, and histologic grade were univariate significant factors associated with a worse MFS. In multivariate analysis, tumor size and tumor grade remained significant prognostic factors for DSS and MFS. A role of SYT-SSX fusion type could not be confirmed in our patient group. Ezrin showed high expression in glandular and nonglandular epithelioid components in biphasic synovial sarcoma. Variable expression was found in the mesenchymal component of monophasic and biphasic synovial sarcoma. Low versus high ezrin expression levels in monophasic and/or biphasic synovial sarcoma did not correlate with patient outcome.
Conclusions: Disease stage at presentation, tumor size, and tumor grade were significant predictors of survival in synovial sarcoma. SYT-SSX fusion type was not correlated with survival in our series. Ezrin expression levels were not discriminative in predicting outcome.
(C) 2009 Lippincott Williams & Wilkins, Inc.
Context: Despite the recognition of the follicular variant of papillary carcinoma of the thyroid (FVPTC) for over 50 years, reproducibility of this diagnostic category has remained poor. Architectural features have been of variable utility as some FVPTC seem encapsulated, whereas others are multifocal and may be confused with nodular hyperplasia. Nuclear features are important for diagnosis of FVPTC, but some authors have discounted the utility of nuclear grooves and inclusions. More recently, BRAF and HBME-1 (Human Bone Marrow Endothelial Cell-1) have been suggested as markers for FVPTC.
Objective: To investigate the frequency of BRAF mutations and HBME-1 immunopositivity, in a series of FVPTCs in which the diagnosis was established by 100% consensus among a panel of 6 surgical pathologists.
Design: Twenty-eight specimens with an original diagnosis of FVPTC and 10 cases with other diagnoses were obtained from the surgical pathology files of the University of Utah School of Medicine. All specimens were independently reviewed by 6 surgical pathologists. Tissue blocks were analyzed for BRAF exon 15 mutations and HMBE-1 expression.
Results: Complete agreement among pathologists for the diagnosis of FVPTC was obtained in 28.6% (8/28) of cases originally diagnosed as FVPTC. Mutations in BRAF exon 15 were found in 25% (2/8) of cases with a 100% consensus diagnosis of FVPTC and 32% (6/19) of cases unanimously diagnosed as a type of papillary carcinoma (classic or follicular variant). HBME-1 was expressed in 87.5% (7/8) of lesions with a 100% consensus diagnosis of FVPTC and 84.2% (16/19) of lesions with a unanimous diagnosis of a type of papillary carcinoma of the thyroid (classic or follicular variant).
Conclusions: Interobserver agreement for the diagnosis of FVPTC is poor and testing for the BRAF mutation is only marginally helpful because a minority of FVPTCs possess the mutation. HBME-1 expression when coupled with a BRAF mutation, results in 100% specificity but low sensitivity for the presence of papillary carcinoma of the thyroid including the follicular variant.
(C) 2010 Lippincott Williams & Wilkins, Inc.
Objectives: Our major hypothesis for these studies was that tamoxifen's varied effects on the endometrium might be due in part to differences in effect on estrogen and progesterone receptors [ER, progesterone receptor isoform A (PRA), and progesterone receptor isoform B (PRB)]. We aimed to evaluate the changes in histology in serial endometrial biopsies (Em bx), Papanicolaou smears (Pap smears), and endometrial ultrasounds as well as changes in the expression of ER, PRA, and PRB in response to tamoxifen. We propose that understanding and correlating the dynamics of receptor expression with histologic and cytologic changes will help us better understand the effect of tamoxifen on the endometrium and its role in the development of endometrial carcinoma in some patients.
Methods: Forty-two patients to be started on tamoxifen underwent a pretreatment Em bx and Pap smear. Follow-up serial Em bxs and Pap smears were obtained at sixth month and then at yearly intervals for up to 6 biopsies per case. Maturation indices (MIs) were determined on the Pap smears, and ER, PRA, and PRB immunostains were performed on the biopsies. Follow-up data is for a maximum of 10 years. Trends in changes in endometrial histology were analyzed and when atrophic or inactive endometrium changed to proliferative endometrium on treatment it was considered to be an increase in estrogen effect and the vice versa changes as a decrease in estrogen effect.
Results: None of the subjects developed hyperplasia or malignancy. Two patients' Em bx demonstrated atypical cells associated with eosinophilic metaplasia, but subsequent biopsies had no atypia. Of the 42 patients, 37 had serial Em bxs in which evaluation for trends could be performed. Twelve of 37 (32.4%) had an overall decrease in estrogen effect on endometrial histology with another 12/37 (32.4%) showing no estrogenic effect on endometrial histology. Six of 37 patients (16.2%) showed an increased estrogen effect on endometrial histology. Seven of 37 (18.9%) had variable endometrial histology with no definable pattern. There was a statistically significant increase in PRA expression compared with baseline as time progressed (P<0.05). The PRB showed a contrasting significant decrease in expression at 2.5 and 3.5 years (P<0.05). There was no significant change in ER expression over the course of the study (P>0.05). Seven of 12 (58.3%) with a decreased estrogenic effect on endometrial histology had a concordant decrease in PRB expression. Seven of 12 (58.3%) with no change in endometrial histology also had a concordant decrease in PRB expression. Comparing the MI of Pap smears with histologic activity of the endometrium revealed minimal correlation between the two. However, in the patients with an increased estrogen effect on endometrial histologic activity, there was no correlation with the MI. Additionally, 57% of patients showed no correlation between endometrial histologic activity and ultrasound findings.
Conclusions: Tamoxifen had an antiestrogenic or neutral effect on endometrial histology and Pap smears of most subjects, but estrogenic, or variable effects were also observed in a minority of patients. Tamoxifen treatment was accompanied by an uncoupling of the regulation of PRA and PRB expression without effect on ER expression. Overall, expression of PRB decreased whereas that of PRA increased.
(C) 2007 Lippincott Williams & Wilkins, Inc.
Introduction: Androgen receptors (AR) have been reported to be present in normal breast tissue and breast carcinomas. The following study was undertaken to assess the expression of AR in vascular neoplasms of the breast.
Materials and Methods: All patients with histologically diagnosed hemangioma, angiolipoma, atypical vascular proliferation, and angiosarcoma of the breast were retrieved from the clinical and pathology database at Rush University Medical Center. The slides stained with hematoxylin and eosin were reviewed and immunohistochemical staining for AR (Dako; clone AR441) was performed on paraffin-embedded tissue. Any amount of nuclear staining was considered positive and the intensity of staining was graded as 1+ to 3+. An estimate of the percentage of tumor and stromal cells staining was made.
Results: There were a total of 36 cases, 10 hemangiomas, 20 angiolipomas, 2 atypical vascular proliferation, and 4 angiosarcomas. The male to female ratio was 1:4.1. The average age at presentation for men was 45 years and for women was 56.9 years. Anti-AR expression was present in stromal and adipocyte nuclei of the angiolipomas and stromal cells of hemangiomas and angiosarcomas. Interestingly, normal duct epithelium of the breast was positive in 7 of the 29 women and none in men. Androgen expression was present in 62.5% of the hemangiomas, 55% of the angiolipomas, and 50% of the angiosarcomas. The majority of tumors showed a low-intensity nuclear expression of androgens, with 1+ intensity in 13 cases and 2+ intensity seen in 2 cases, and only 1 case of angiolipoma showed 3+ expression. All positive cases of angiolipoma (55%) showed AR in adipocytes and stromal cells.
Conclusions: Vascular neoplasms are uncommon tumors in breast and majority of them are benign. Androgen expression is present in >50% of benign vascular neoplasms. Stromal cells and adipocytes typically express AR only in angiolipomas, suggesting a role of androgens in pathogenesis and growth of this neoplasm.
(C) 2013 Lippincott Williams & Wilkins, Inc.
Losses in the succinate dehydrogenase (SDH) complex characterize 20% to 30% of extra-adrenal paragangliomas and 7% to 8% of gastric GISTs, and rare renal cell carcinomas. This loss is reflected as lack of the normally ubiquitous immunohistochemical expression of the SDH subunit B (SDHB). In paragangliomas, SDHB loss correlates with homozygous loss of any of the SDH subunits, typically by loss-of-function mutations. The occurrence of SDHB losses in other epithelial malignancies is unknown. In this study, we immunohistochemically examined 2258 epithelial, mostly malignant neoplasms including common carcinomas of all sites. Among renal cell carcinomas, SDHB loss was observed in 4 of 711 cases (0.6%), including a patient with an SDHB-deficient GIST. Histologically, the SDHB-negative renal carcinomas varied. There was 1 clear cell carcinoma with a high nuclear grade, 1 papillary carcinoma type 2, 1 unclassified carcinoma with a glandular pattern, and 1 oncocytoid low-grade carcinoma as previously described for SDHB-negative renal carcinoma. None of these patients was known to have paragangliomas or had loss of SDHA expression in the tumor. Three of these patients had metastases at presentation (2 in the adrenal, 1 in the retroperitoneal lymph nodes). There were no cases with SDHB loss among 64 renal oncocytomas. SDHB losses were not seen in other carcinomas, except in 1 prostatic adenocarcinoma (1/57), 1 lymphoepithelial carcinoma of the stomach, and 1 (1/40) seminoma. On the basis of this study, SDHB losses occur in 0.6% of renal cell carcinomas and extremely rarely in other carcinomas. Some of these renal carcinomas may be clinically aggressive. The clinical significance and molecular genetics of these SDHB-negative tumors requires further study.
(C) 2013 Lippincott Williams & Wilkins, Inc.
Survivin is an inhibitor of apoptosis protein that inhibits caspase 3 function. While cytoplasmic survivin suppresses apoptosis, nuclear survivin regulates cell division. Little is known about the subcellular localization of survivin in oral carcinogenesis. This study examined the subcellular distribution of these 2 proteins in oral squamous cell carcinoma (OSCC) and premalignant lesions including oral leukoplakia (OL) with and without dysplasia. Expression of survivin and caspase 3 were immunohistochemically analyzed in 114 samples including OSCC, OL with and without dysplasia, and normal oral mucosa (NM). Cytoplasmic and nuclear positive cells were counted separately. The results were presented as the frequency of positive cases. The positive expression rates of cytoplasmic and nuclear survivin in OSCC were significantly higher than in NM, OL with and without dysplasia. NM showed a low rate of cytoplasmic survivin expression compared to OL with and without dysplasia. The numbers of cytoplasmic and nuclear expression of caspase 3 in OSCC were significantly higher than that of NM, OL with and without dysplasia. In conclusion, the overexpression of cytoplasmic survivin in OSCC and premalignant lesions suggest that suppression of apoptosis by survivin occurs at early and late stages of oral carcinogenesis. The elevated expression of nuclear survivin and caspase 3 in OSCC indicate that at the late stage survivin increases cell proliferation whereas caspase 3 promotes apoptosis.
(C) 2013 Lippincott Williams & Wilkins, Inc.
Sorting nexins (SNXs) are a large, diverse group of cytoplasmic and membrane-associated proteins that function in a variety of cellular processes, including endocytosis, protein trafficking, and the retrieval of transmembrane proteins. In this study, we demonstrated that SNX2 is expressed in columnar and active thyroid follicular cells but not in flattened inactive thyrocytes. Morphometric analysis revealed a significant correlation between SNX2 positivity and columnar cell morphology. Immunohistochemical staining of serial sections of the thyroid tissue indicated that SNX2 localization was similar to sortilin, a protein expressed by active thyrocytes. Expression of SNX2 in thyrocytes is particularly marked and extensive in most hyperstimulated thyroid disorders, including Graves disease (diffusely SNX2 positive in 73.3% patients) and functioning nodules (93.8% patients). SNX2 immunolocalization in hyperstimulated follicular epithelial cells was specific among the SNXs family members examined. These results support the utility of SNX2 as a novel marker of active thyrocytes and reflect the endosomal trafficking activity in these cells.
(C) 2013 Lippincott Williams & Wilkins, Inc.
Manual tissue microarray (TMA) construction had been introduced to avoid the high cost of automated and semiautomated techniques. The cheapest and simplest technique for constructing manual TMA was that of using mechanical pencil tips. This study was carried out to modify this method, aiming to raise its quality to reach that of expensive ones. Some modifications were introduced to Shebl’s technique. Two conventional mechanical pencil tips of different diameters were used to construct the recipient blocks. A source of mild heat was used, and blocks were incubated at 38°C overnight. With our modifications, 3 high-density TMA blocks were constructed. We successfully performed immunostaining without substantial tissue loss. Our modifications increased the number of cores per block and improved the stability of the cores within the paraffin block. This new, modified technique is a good alternative for expensive machines in many laboratories.
Background:A recently described mucinous non-neoplastic cyst (MNNC) of the pancreas is a benign cyst and should be distinguished from mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm (IPMN) due to different management and prognosis. The immunoprofile of MNNC has not been well studied.
Design:Twenty-three MNNCs diagnosed on surgical resection were retrieved. Immunohistochemical (IHC) staining was performed on surgically resected specimen. Sixteen IPMN and 15 MCN cases were also retrieved for comparison. Cyst fluid carcinoembryonic antigen and amylase concentrations were retrieved.
Result:MNNCs were randomly located in the pancreas and were either unilocular or multilocular cysts that were lined by a single layer of bland columnar or cuboidal mucinous cells and supported by paucicellular stroma. The glandular epithelial cells were diffusely positive for CK7 (100%) and PDX-1 (65%); focally positive for CD10 (superficial, 65%), CD99 (basally, 100%), CDX-2 (17%), and CK20 (4%); and negative for MUC2. Only rare stromal cells in the cyst wall were weakly positive for estrogen receptor or progesterone receptor in only 6% of cases and negative for inhibin. These results were also compared with the immunoprofile of IPMN and MCN.
Conclusions:Although MNNC shares some IHC markers with IPMN or MCN, an IHC panel can help distinguish MNNC from IPMN or MCN. The results suggest that MNNC is different from IPMN and MCN.
Introduction:Glutamine synthetase (GS), heat shock protein-70 (HSP-70), and glypican-3 (GPC-3) are markers best characterized in hepatocellular lesions, where they are useful in distinguishing hepatocellular carcinoma from dysplastic nodules. Their staining patterns in intrahepatic cholangiocarcinoma (IH-ChCa) and metastatic tumors in liver are not well described.
Methods:Tissue microarrays containing 41 IH-ChCa and 24 metastatic tumors in liver were stained with commercially available antibodies to GS, HSP-70, and GPC-3. Five percent staining of tumor cells was considered positive for HSP-70 and GPC-3. For GS, 50% was the cut-off.
Results:GS reactivity was present in 31 of 41 IH-ChCa (76%), with the median amount of staining being 65% of tumor cells. HSP-70 reactivity was present in 36 of 41 IH-ChCa (88%) with the median amount of staining being 75% of tumor cells. GPC-3 reactivity was absent from all IH-ChCa. Twenty-seven of 41 IH-ChCa cases were positive for both GS and HSP-70 (66%). GS reactivity was present in 17 of 24 tumors metastatic to liver (71%), with the median amount of staining being 50% of tumor cells. HSP-70 reactivity was present in 21 of 24 tumors metastatic to liver (88%) with the median amount of staining being 80% of tumor cells. GPC-3 reactivity was present in 2 of 24 tumors metastatic to liver (8%) with one showing 5% staining and the other showing 50% staining of tumor cells. Fifteen of 24 cases were positive for both GS and HSP-70 (63%), and 2 cases were positive for all 3 markers (8%).
Discussion:Of the panel of immunostains currently commonly used to distinguish hepatocellular carcinoma from dysplastic hepatocytic nodules, only GPC-3 did not react frequently with metastatic tumors and IH-ChCa, although there was staining in 2 metastatic tumors. GS and HSP-70 are typically positive in IH-ChCa and metastatic tumors. Nothing should be inferred about the histogenesis of a tumor based on positive staining with either of these 2 markers, which currently have no role in tumor of unknown origin panels.
Melanins are naturally occurring pigments in both normal and pathologic tissues. Two common bleaching processes are potassium permanganate followed by oxalic acid treatment and dilute hydrogen peroxide (H2O2) process. The potassium permanganate/oxalic acid method is faster and more easily incorporated in conventional daily immunostaining protocols, whereas the dilute H2O2 method requires 24 hours. This study aimed to reduce melanin bleaching time by using a 10% H2O2 dilution. First, reaction time was reduced to 30 minutes by raising the temperature to 65°C. Second, containers with high thermal conductivity were used to improve bleaching effectiveness. Experimental comparisons of melanin treatments with H2O2 contained in an iron jar, a glass coplin jar, and a plastic steel jar obtained bleaching time of 20, 30, and 40 minutes, respectively. These modifications of the conventional bleaching method significantly improve the speed and efficiency of the procedure and are recommended when performing immunohistochemical studies.
The endothelial barrier controls the passage of fluids, nutrients and cells through the vascular wall. This physiological function is closely related to developmental and adult angiogenesis, blood pressure control, as well as immune responses. Moreover, cancer progression is frequently characterized by disorganized and leaky blood vessels. In this context, vascular permeability drives tumour-induced angiogenesis, blood flow disturbances, inflammatory cell infiltration and tumour cell extravasation. Although various molecules have been implicated, the vascular endothelial adhesion molecule, VE-cadherin (vascular endothelial cadherin), has emerged as a critical player involved in maintaining endothelial barrier integrity and homoeostasis. Indeed, VE-cadherin coordinates the endothelial cell-cell junctions through its adhesive and signalling properties. Of note, many angiogenic and inflammatory mediators released into the tumour microenvironment influence VE-cadherin behaviour. Therefore restoring VE-cadherin function could be one very promising target for vascular normalization in cancer therapies. In this review, we will mainly focus on recent discoveries concerning the molecular mechanisms involved in modulating VE-cadherin plasticity in cancer.
Integrating signals from the ECM (extracellular matrix) via the cell surface into the nucleus is an essential feature of multicellular life and often malfunctions in cancer. To date many signal transducers known as shuttle proteins have been identified that act as both: a cytoskeletal and a signalling protein. Here, we highlight the interesting member of the Zyxin family TRIP6 [thyroid receptor interactor protein 6; also designated ZRP-1 (zyxin-related protein 1)] and review current literature to define its role in cell physiology and cancer. TRIP6 is a versatile scaffolding protein at FAs (focal adhesions) involved in cytoskeletal organization, coordinated cell migration and tissue invasion. Via its LIM and TDC domains TRIP6 interacts with different components of the LPA (lysophosphatidic acid), NF-κB (nuclear factor κB), glucocorticoid and AMPK (AMP-activated protein kinase) signalling pathway and thereby modulates their activity. Within the nucleus TRIP6 acts as a transcriptional cofactor regulating the transcriptional responses of these pathways. Moreover, intranuclear TRIP6 associates with proteins ensuring telomere protection and hence may contribute to genome stability. Accordingly, TRIP6 is engaged in key cellular processes such as cell proliferation, differentiation and survival. These diverse functions of TRIP6 are found to be dysregulated in various cancers and may have pleiotropic roles in tumour initiation, tumour growth and metastasis, which turn TRIP6 into an attractive candidate for cancer diagnosis and targeted therapy.
Background information. Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H+,K+ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells.
Results. Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H+,K+ ATPase-deficient mice that lack tubulovesicular membranes.
Conclusions. These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.
Background:
The second-order, infinite impulse response notch filter is widely used to remove electrical power line noise in electrocardiograms (ECGs). However this filtering process often introduces spurious ringing artifacts in the vicinity of raw signal with sharp transitions. It is challenging to simultaneously remove these two types of noise without losing vital information about cardiac activities.ObjectiveOur objective is to devise a method to remove the power-line interference without introducing artifacts nor losing vital information. To this end we have developed the "hybrid approach" involving two-sided filtration and multi-iterative approximation techniques. The two-sided filtration technique can suppress the interference but some cardiac components are lost. The lost information can be restored using multi-iterative approximation technique.
Results:
For evaluation, four artificial data sets, each including 91 ECGs of different heart rates, were generated by a dynamical model. Four publicly-accessible sets of clinical data (MIT-BIH Arrhythmia, QT, PTB Diagnostic ECG, and T-Wave Alternans Challenge Databases) were also selected. Our new hybrid approach and the existing method were tested with these two types of signal under various pre-determined conditions. In contrast with the existing method, the hybrid approach can provide more than 27.40 dB and 37.77 dB reduction in signal distortion for 95% and 60% of artificial ECGs respectively; it can provide in excess of 11.78 dB and 17.48 dB reduction in distortion for 95% and 60% of these real records respectively.
Conclusions:
Overall, a significant reduction in signal distortion is demonstrated. These test results indicate that the newly proposed approach outperforms the traditional method assessed on both the artificial and clinical ECGs and suggest it could be of practical use for clinicians in the future.
IntroductionThis paper presents the problem of automatic measurement of the iridocorneal angle in tomographic images of the anterior segment of the eye. It includes the results of the comparison of well-known methods for measuring the iridocorneal angle with new methods, proposed in this paper. All these methods concern tomographic image analysis and processing.Material and methodIn total, approximately 100'000 tomographic images (from about 6'000 patients) were analysed. They were obtained using two devices: SOCT Copernicus (Optopol Tech. SA, Zawiercie, Poland) and Visante OCT (Carl Zeiss Meditec, Inc, Dublin, California, USA). The patients, aged 12 to 78 years with varying degrees of the iridocorneal angle pathology, were from the region of Silesia, Poland. The images were in DICOM or RAW formats and analysed in the software developed by the authors for the purposes of this study.
Results:
The results indicate that the measurement method proposed by the authors, which is based on the calculation of the minimum distance between the iris and the cornea in the adopted area, is the most accurate. For this method sensitivity was 0.88, specificity 0.89 and the area under the Receiver Operating Characteristic curve (AUC) was 0.88. The other known methods for measuring the iridocorneal angle gave worse results, that is, for example, for the measurement of the distance between the iris and the cornea AUC = 0.87, sensitivity = 0.86 and specificity = 0.71. For another well-known method of measuring the iridocorneal angle AUC = 0.77, sensitivity = 0.82 and specificity = 0.61.
Conclusions:
The study proved that the proposed method of measuring the minimum distance between the iris and the cornea within the adopted area is the most effective in the classification of the iridocorneal angle in patients with a high degree of pathology of all the compared measurement methods based on tomographic images. However, it requires fully automated measurement.
Background:
The fovea, which is the most sensitive part of the retina, is known to have birefringent properties, i.e. it changes the polarization state of light upon reflection. Existing devices use this property to obtain information on the orientation of the fovea and the direction of gaze. Such devices employ specific frequency components that appear during moments of fixation on a target. To detect them, previous methods have used solely the power spectrum of the Fast Fourier Transform (FFT), which, unfortunately, is an integral method, and does not give information as to where exactly the events of interest occur. With very young patients who are not cooperative enough, this presents a problem, because central fixation may be present only during very short-lasting episodes, and can easily be missed by the FFT.MethodThis paper presents a method for detecting short-lasting moments of central fixation in existing devices for retinal birefringence scanning, with the goal of a reliable detection of eye alignment. Signal analysis is based on the Continuous Wavelet Transform (CWT), which reliably localizes such events in the time-frequency plane. Even though the characteristic frequencies are not always strongly expressed due to possible artifacts, simple topological analysis of the time-frequency distribution can detect fixation reliably.
Results:
In all six subjects tested, the CWT allowed precise identification of both frequency components. Moreover, in four of these subjects, episodes of intermittent but definitely present central fixation were detectable, similar to those in Figure 4. A simple FFT is likely to treat them as borderline cases, or entirely miss them, depending on the thresholds used.
Conclusion:
Joint time-frequency analysis is a powerful tool in the detection of eye alignment, even in a noisy environment. The method is applicable to similar situations, where short-lasting diagnostic events need to be detected in time series acquired by means of scanning some substrate along a specific path.
Background:
Intravascular ultrasound (IVUS) is a standard imaging modality for identification of plaque formation in the coronary and peripheral arteries. Volumetric three-dimensional (3D) IVUS visualization provides a powerful tool to overcome the limited comprehensive information of 2D IVUS in terms of complex spatial distribution of arterial morphology and acoustic backscatter information. Conventional 3D IVUS techniques provide sub-optimal visualization of arterial morphology or lack acoustic information concerning arterial structure due in part to low quality of image data and the use of pixel-based IVUS image reconstruction algorithms. In the present study, we describe a novel volumetric 3D IVUS reconstruction algorithm to utilize IVUS signal data and a shape-based nonlinear interpolation.
Methods:
We developed an algorithm to convert a series of IVUS signal data into a fully volumetric 3D visualization. Intermediary slices between original 2D IVUS slices were generated utilizing the natural cubic spline interpolation to consider the nonlinearity of both vascular structure geometry and acoustic backscatter in the arterial wall. We evaluated differences in image quality between the conventional pixel-based interpolation and the shape-based nonlinear interpolation methods using both virtual vascular phantom data and in vivo IVUS data of a porcine femoral artery. Volumetric 3D IVUS images of the arterial segment reconstructed using the two interpolation methods were compared.
Results:
In vitro validation and in vivo comparative studies with the conventional pixel-based interpolation method demonstrated more robustness of the shape-based nonlinear interpolation algorithm in determining intermediary 2D IVUS slices. Our shape-based nonlinear interpolation demonstrated improved volumetric 3D visualization of the in vivo arterial structure and more realistic acoustic backscatter distribution compared to the conventional pixel-based interpolation method.
Conclusions:
This novel 3D IVUS visualization strategy has the potential to improve ultrasound imaging of vascular structure information, particularly atheroma determination. Improved volumetric 3D visualization with accurate acoustic backscatter information can help with ultrasound molecular imaging of atheroma component distribution.
Optical imaging techniques reflect different biochemical processes in the brain, which is closely related with neural activity. Scientists and clinicians employ a variety of optical imaging technologies to visualize and study the relationship between neurons, glial cells and blood vessels. In this paper, we present an overview of the current optical approaches used for the in vivo imaging of neurovascular coupling events in small animal models. These techniques include 2-photon microscopy, laser speckle contrast imaging (LSCI), voltage-sensitive dye imaging (VSDi), functional photoacoustic microscopy (fPAM), functional near-infrared spectroscopy imaging (fNIRS) and multimodal imaging techniques. The basic principles of each technique are described in detail, followed by examples of current applications from cutting-edge studies of cerebral neurovascular coupling functions and metabolic. Moreover, we provide a glimpse of the possible ways in which these techniques might be translated to human studies for clinical investigations of pathophysiology and disease. In vivo optical imaging techniques continue to expand and evolve, allowing us to discover fundamental basis of neurovascular coupling roles in cerebral physiology and pathophysiology.
Background:
The improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG).
Results:
In biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer.
Conclusions:
Thus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.
Background:
The archeaon Sulfolobus solfataricus P2 encodes a thermoacidophilic cellulase which shows an extreme acid and thermal stability with a pH optimum at 1.8 and a temperature optimum at 80[degree sign]C. This extraordinary enzyme could be useful for biotechnological exploitation but the expression and purification in expression hosts like E. coli is unsatisfactory due to the high aggregation tendency of the recombinant enzyme. The thermophilic cellulase CelA from Thermotoga maritima belongs to the same glycoside hydrolase family (GH12) but has a neutral pH optimum. In contrast to SSO1949 this enzyme is expressed partially soluble in E. coli.
Results:
We aimed to constructed a hybrid enzyme based on these two beta-endoglucanases which should successfully combine the advantageous properties of both cellulases, i.e. recombinant expression in E. coli, acidophily and thermophily. We constructed two hybrid proteins after bioinformatic analysis: both hybrids are expressed insoluble in E. coli, but one hybrid enzyme was successfully refolded from washed inclusion bodies.
Conclusions:
The refolded active chimeric enzyme shows a temperature optimum of approximately 85[degree sign]C and a pH optimum of approximately pH 3 thus retaining the advantageous properties of the Sulfolobus parent enzyme. This study suggests that the targeted construction of chimeric enzymes is an alternative to point mutational engineering efforts as long as parent enzymes with the wanted properties are available.
Background:
Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464--5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor.
Results:
Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1--3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5.
Conclusions:
Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high-affinity interaction involving an antibody from the VH1-69 germline segment.
Background:
The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily of ligand-inducible DNA transcription factors, and is the major mediator of male sexual development, prostate growth and the pathogenesis of prostate cancer. Cell and gene specific regulation by the AR is determined by availability of and interaction with sets of key accessory cofactors. Ski-interacting protein (SKIP; SNW1, NCOA62) is a cofactor shown to interact with several NRs and a diverse range of other transcription factors. Interestingly, SKIP as part of the spliceosome is thought to link mRNA splicing with transcription. SKIP has not been previously shown to interact with the AR.
Results:
The aim of this study was to investigate whether SKIP interacts with the AR and modulates AR-dependent transcription. Here, we show by co-immunoprecipitation experiments that SKIP is in a complex with the AR. Moreover, SKIP increased 5alpha-dihydrotestosterone (DHT) induced N-terminal/C-terminal AR interaction from 12-fold to almost 300-fold in a two-hybrid assay, and enhanced AR ligand-independent AF-1 transactivation. SKIP augmented ligand- and AR-dependent transactivation in PC3 prostate cancer cells. Live-cell imaging revealed a fast (half-time=129 s) translocation of AR from the cytoplasm to the nucleus upon DHT-stimulation. Forster resonance energy transfer (FRET) experiments suggest a direct AR-SKIP interaction in the nucleus upon translocation.
Conclusions:
Our results suggest that SKIP interacts with AR in the nucleus and enhances AR-dependent transactivation and N/C-interaction supporting a role for SKIP as an AR co-factor.
Contributing reviewersThe editors of BMC Biochemistry would like to thank all of our reviewers who have contributed to the journal in volume 13 (2012).
Background:
In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies ("D1.3") produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used.
Results:
Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments.
Conclusions:
We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps.
Background:
Plant expansins and fungal swollenin that can disrupt crystalline cellulose have great potential for applications in conversion of biomass. Recent studies have been mainly focused on Trichoderma reesei swollenin that show relatively low activity in the promotion of cellulosic hydrolysis. Our aim was to isolate a novel swollenin with greater disruptive activity, to establish an efficient way of producing recombinant swollenin, and to optimize the procedure using swollenin in facilitation of cellulosic hydrolysis.
Results:
A novel gene encoding a swollenin-like protein, POSWOI, was isolated from the filamentous fungus Penicillium oxalicum by Thermal Asymmetric Interlaced PCR (TAIL-PCR). It consisted of a family 1 carbohydrate-binding module (CBM1) followed by a linker connected to a family 45 endoglucanase-like domain. Using the cellobiohydrolase I promoter, recombinant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/mL, and showed significant disruptive activity on crystalline cellulose. Simultaneous reaction with both POSWOI and cellulases enhanced the hydrolysis of crystalline cellulose Avicel by approximately 50%. Using a POSWOI-pretreatment procedure, cellulases can produce nearly twice as many reducing sugars as without pretreatment. The mechanism by which POSWOI facilitates the saccharification of cellulose was also studied using a cellulase binding assay.
Conclusion:
We present a novel fungal swollenin with considerable disruptive activity on crystalline cellulose, and develop a better procedure for using swollenin in facilitating cellulosic hydrolysis. We thus provide a new approach for the effective bioconversion of cellulosic biomass.
Background:
Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency.
Results:
109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring.
Conclusion:
Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.
Background:
The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored.
Results:
The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes.
Conclusions:
We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.
Background:
Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptides (ELP), hydrophobin (HFBI) and Zera(R) are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum.
Results:
The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants.
Conclusion:
The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.
Background:
Notch signaling, a critical pathway for tissue development, contributes to tumorigenesis in many tissues; however, the roles of Notch signaling in Intrahepatic Cholangiocarcinoma (ICC) remains unclear. In this study, we evaluated the expression and effects of Notch1 on cell migration in ICC.
Methods:
Multiple cellular and molecular approaches were performed including gene transfection, siRNA transfection, RT-PCR, Western blotting, Rac activation assays and immunofluorescence.
Results:
We found that Notch1 was up-regulated in ICC tissues and cell lines. The exogenous expression of Notch1 in glioma cells increased their migratory and invasive capacity. Similarly, the suppression of Notch1 expression inactivated Rac1 and inhibited ICC cell migration. Notch1 over expression induced an Epithelial-to-mesenchymal transition (EMT) phenotype that included enhanced expression of alpha-SMA and Vimentin, loss of E-cadherin expression, morphological changes and cytoskeletal reorganization in ICC cells.
Conclusion:
Notch1 may induce a migratory effect in ICC by causing an epithelial-mesenchymal transition and activating Rac1 and could serve as a novel diagnostic and therapeutic target in patients with ICC.
Background:
Evidence suggests that cytoglobin (Cygb) may function as a tumor suppressor gene.
Methods:
We immunohistochemically evaluated the expression of Cygb, phosphatidylinositol-3 kinase (PI-3K), phosphorylated (p)-Akt, Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFalpha) and vascular endothelial growth factor (VEGF) in 88 patients with 41 high-grade gliomas and 47 low-grade gliomas. Intratumoral microvessel density (IMD) was also determined and associated with clinicopathological factors.
Results:
Low expression of Cygb was significantly associated with the higher histological grading and tumor recurrence. A significant negative correlation emerged between Cygb expression and PI3K, p-Akt, IL-6, TNFalpha or VEGF expression. Cygb expression was negatively correlated with IMD. There was a positive correlation between PI3K, p-Akt, IL-6, TNFalpha and VEGF expression with IMD.High histologic grade, tumor recurrence, decreased Cygb expression, increased PI3K expression, increased p-Akt expression and increased VEGF expression correlated with patients' overall survival in univariate analysis. However, only histological grading and Cygb expression exhibited a relationship with survival of patients as independent prognostic factors of glioma by multivariate analysis.
Conclusions:
Cygb loss may contribute to tumor recurrence and a worse prognosis in gliomas. Cygb may serve as an independent predictive factor for prognosis of glioma patients.
Background:
High-grade osteosarcoma is an aggressive tumor most often developing in the long bones of adolescents, with a second peak in the 5th decade of life. Better knowledge on cellular signaling in this tumor may identify new possibilities for targeted treatment.
Methods:
We performed gene set analysis on previously published genome-wide gene expression data of osteosarcoma cell lines (n=19) and pretreatment biopsies (n=84). We characterized overexpression of the insulin-like growth factor receptor (IGF1R) signaling pathways in human osteosarcoma as compared with osteoblasts and with the hypothesized progenitor cells of osteosarcoma -- mesenchymal stem cells. This pathway plays a key role in the growth and development of bone. Since most profound differences in mRNA expression were found at and upstream of the receptor of this pathway, we set out to inhibit IR/IGF1R using OSI-906, a dual inhibitor for IR/IGF1R, on four osteosarcoma cell lines. Inhibitory effects of this drug were measured by Western blotting and cell proliferation assays.
Results:
OSI-906 had a strong inhibitory effect on proliferation of 3 of 4 osteosarcoma cell lines, with IC50s below 100nM at 72 hrs of treatment. Phosphorylation of IRS-1, a direct downstream target of IGF1R signaling, was inhibited in the responsive osteosarcoma cell lines.
Conclusions:
This study provides an in vitro rationale for using IR/IGF1R inhibitors in preclinical studies of osteosarcoma.
Background:
The Follicle Stimulating Hormone receptor (FSHR) is expressed by the vascular endothelium in a wide range of human tumors. It was not determined however if FSHR is present in metastases which are responsible for the terminal illness.
Methods:
We used immunohistochemistry based on a highly FSHR-specific monoclonal antibody to detect FSHR in cancer metastases from 6 major tumor types (lung, breast, prostate, colon, kidney, and leiomyosarcoma ) to 6 frequent locations (bone, liver, lymph node, brain, lung, and pleura) of 209 patients.
Results:
In 166 patients examined (79%), FSHR was expressed by blood vessels associated with metastatic tissue. FSHR-positive vessels were present in the interior of the tumors and some few millimeters outside, in the normally appearing tissue. In the interior of the metastases, the density of the FSHR-positive vessels was constant up to 7 mm, the maximum depth available in the analyzed sections. No significant differences were noticed between the density of FSHR-positive vessels inside vs. outside tumors for metastases from lung, breast, colon, and kidney cancers. In contrast, for prostate cancer metastases, the density of FSHR-positive vessels was about 3-fold higher at the exterior of the tumor compared to the interior. Among brain metastases, the density of FSHR-positive vessels was highest in lung and kidney cancer, and lowest in prostate and colon cancer. In metastases of breast cancer to the lung pleura, the percentage of blood vessels expressing FSHR was positively correlated with the progesterone receptor level, but not with either HER-2 or estrogen receptors. In normal tissues corresponding to the host organs for the analyzed metastases, obtained from patients not known to have cancer, FSHR staining was absent, with the exception of approx. 1% of the vessels in non tumoral temporal lobe epilepsy samples.
Conclusion:
FSHR is expressed by the endothelium of blood vessels in the majority of metastatic tumors.
Background:
Chemotherapy with trastuzumab is widely used for patients with human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but a significant number of patients with the tumor fail to respond, or relapse. The mechanisms of recurrence and biomarkers that indicate the response to the chemotherapy and outcome are not fully investigated.
Methods:
Genomic alterations were analyzed using single-nucleotide polymorphism arrays in 46 HER2 immunohistochemistry (IHC) 3+ or 2+/fluorescent in situ hybridization (FISH)+ breast cancers that were treated with neoadjuvant chemotherapy with paclitaxel, cyclophosphamid, epirubicin, fluorouracil, and trastuzumab. Patients were classified into two groups based on presence or absence of alterations of 65 cancer-associated genes, and the two groups were further classified into four groups based on genomic HER2 copy numbers or hormone receptor status (HR+/-). Pathological complete response (pCR) and relapse-free survival (RFS) rates were compared between any two of the groups.Results and discussionThe pCR rate was 54% in 37 patients, and the RFS rate at 3 years was 72% (95% CI, 0.55-0.89) in 42 patients. The analysis disclosed 8 tumors with nonamplified HER2 and 38 tumors with HER2 amplification, indicating the presence of discordance in tumors diagnosed using current HER2 testing. The 8 patients showed more difficulty in achieving pCR (P=0.019), more frequent relapse (P=0.018), and more frequent alterations of genes in the PI3K pathway (P=0.009) than the patients with HER2 amplification. The alterations of the PI3K and estrogen receptor (ER) pathway genes generally indicated worse RFS rates. The prognostic significance of the alterations was shown in patients with a HR+ tumor, but not in patients with a HR- tumor when divided. Alterations of the PI3K and ER pathway genes found in patients with a HR+ tumor with poor outcome suggested that crosstalk between the two pathways may be involved in resistance to the current chemotherapy with trastuzumab.
Conclusions:
We recommend FISH analysis as a primary HER2 testing because patients with IHC 2+/3+ and nonamplified HER2 had poor outcome. We also support concurrent use of trastuzumab, lapatinib, and cytotoxic and anti-hormonal agents for patients having HR+ tumors with alterations of the PI3K and ER pathway genes.
Background:
We have recently characterized two distinct populations of Satellite Cells (SCs) that differ in proliferation, regenerative potential, and mitochondrial coupling efficiency and classified these in Low Proliferative Clones (LPC) and High Proliferative Clones (HPC). Herewith, we have investigated their cell metabolism and individuated features that remark an intrinsic difference in basal physiology but that are retrievable also at the initial phases of their cloning.
Results:
Indeed, LPC and HPC can be distinguished for mitochondrial membrane potential (DeltaPsim) just after isolation from the fiber. This is matched by mitochondrial redox state measured via NAD+/NADH analysis and alternative respiratory CO2 production in cloned cells. All these parameters are accountable for metabolic differences reflected indeed by alternative expression of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3). Also Ca2+ handling by mitochondria is different together with the sensitivity to apoptosis triggered via this pathway. Finally, according to the above, we were able to determine which one among the clones represents the suitable stem cell.
Conclusions:
These experimental observations report novel physiological features in the cell biology of SCs and refer to an intrinsic heterogeneity within which their stemness may reside.
Background:
Previous studies have implicated continuous or intermittent hyperglycemia in altered endothelium-derived nitric oxide (NO) synthesis. NO can regulate both the F-actin cytoskeleton and endothelial cell membrane stiffness. Atomic force microscopy (AFM) is a powerful tool that can be used to study plasma membrane deformability at the single cell level. As membrane stiffness is partially dependent on filamentous F-actin, the interdependence of these parameters can be studied through the combined approaches of AFM and laser scanning confocal microscopy (LSCM). In the present study, we evaluated the effects of constant or fluctuating hyperglycemia on endothelial-derived NO synthesis, the cytoskeletal contribution and endothelial cell membrane stiffness.
Results:
Compared to control cells cultured in low glucose (5mM), constant (25mM) or fluctuating (25/5mM) high glucose significantly decreased NO release along with stiffening of endothelial cell membranes and F-actin rearrangement. The non-selective nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) exerted similar effects on endothelial cells. Increasing concentrations of L-NAME (from 0.1 to 1 mM) exacerbated these effects in a concentration-dependent manner.
Conclusions:
Result from the present study suggest that stiffening endothelial cell membranes are associated with decreased NO synthesis, which was established through the F-actin cytoskeletal redistribution. The precise mechanisms of hyperglycemia-induced endothelial dysfunction require further investigation.
Background:
The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance.
Results:
MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform.
Conclusions:
Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics but also for the evaluation of treatment regimens and predicting disease outcomes.
Background:
The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19--26 nucleotides in length.
Results:
We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1--2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection.
Conclusions:
Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1--2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence.
Background:
Krabbe disease, also known as globoid cell leukodystrophy, is an autosomal recessive neurodegenerative disease caused by the genetic deficiency of galactocerebrosidase (GALC), a lysosomal enzyme responsible for the degradation of several glycosphingolipids like psychosine and galactosylceramide. In order to investigate whether GALC deficiency in Krabbe disease affects adipose-derived stromal/stem cell (ASC) properties and if the ASCs could be used as a source of autologous stem cell therapy for patients with Krabbe disease, ASCs isolated from subcutaneous adipose tissue of Twitcher mice (a murine model of Krabbe disease) and their normal wild type littermates were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, growth kinetics, and immune regulatory capacities in vitro.
Results:
ASCs from Twitcher mice (TwiASCs), when compared to ASCs from normal mice (WtASCs), have a reduced osteogenic differentiation potential, have less self-replicating and proliferative capacity, although they have the same fibroblast morphologies and cell sizes. However, surprisingly, the TwiASCs demonstrated similar immune-suppressive capacities as their counterparts WtASCs did when they were transwell co-cultured with macrophages in vitro.
Conclusion:
This study reveals that Twitcher ASCs exhibit differences in the biologic potential when compared to their counterparts from normal mice. The changes in Twitcher ASCs may be influenced by the GALC deficiency in Twitcher mice. Nevertheless, none of the changes preclude the use of the TwiASCs for autologous applications.
Background:
Tumor epidemiology is a significant part of CNS (central nervous system) tumor studies. Reassessment of original sections can update our knowledge of tumor spectrum. Here, we discuss the features of CNS tumor pathology in a single center.
Methods:
A total of 34140 cases from 1950 to 2009 were collected; sections from 1990 to 2009 were reassessed according to WHO 2007 classification, and cases from 1950 to 1989 were classified according to the previous pathological diagnosis.
Results:
Seven CNS tumor categories during 1990 to 2009 were as follow: neuroepithelial tissue (38.0%), tumors of the meninges (36.5%), tumors of the sellar region (4.1%), germ cell tumors (1.3%), tumors of cranial and paraspinal nerves (13.3%), lymphomas and hematopoietic neoplasm (1.7%), metastatic tumors (5.1%), where histological types by age and sex were diverse. Overall, males exceeded females in distributions of most CNS tumor subtypes, while tumors of the meninges occurred more frequently in females. The case number of lymphomas and hematopoietic neoplasms grew the fastest during the past five years, and the distribution of neuroepithelial tumors remained stable over the past twenty years.
Conclusions:
Despite the possibilities of cross sample biases, the data in this series could suggest a similar CNS tumor spectrum as might occur in other developing countries.
Background:
Positive HER2 status identifies breast carcinomas that might respond to trastuzumab treatment. Manual HER2 fluorescent in situ hybridisation (FISH) is the most readily used method to detect HER2 gene amplification which defines positive HER2 status in addition to HER2 protein overexpression. Automation of HER2 FISH may improve HER2 gene testing. The aim of our study was to evaluate an automated HER2 FISH assay for assessing the HER2 genomic status.
Methods:
Core biopsies of 100 invasive breast carcinomas were analysed in parallel using the PathVysionTM HER-2 DNA Probe Kit and the Leica HER2 FISH System for BONDTM. To assess inter-method agreement, concordance analysis was performed for various numerical and categorical HER2/CEP17 FISH parameters.
Results:
Carcinomas with all HER2 immunohistochemical scores were included (0+: 20; 1+: 20; 2+: 30; 3+: 30). Using either HER2/CEP17 ratio >2.2 or >=2.0 as criterion for HER2 amplification, high levels of concordance were observed between automated and manual FISH (concordance rate 96%, Kappa coefficient 0.92). High levels of inter-method agreement were also found for HER2 copy number, CEP17 copy number, HER2/CEP17 ratio, the percentage of carcinoma cells with HER2/CEP17 ratio >2.2, and the presence of HER2 genetic heterogeneity, HER2 clusters and CEP17 polyploidy.
Conclusions:
HER2 testing using automated FISH is feasible on breast carcinoma core biopsies. Automated HER2 FISH using the Leica HER2 FISH System for BOND is a practical and efficient alternative to manual HER2 FISH in evaluating the HER2 status of primary invasive breast carcinomas.
Background:
Serum albumin is a marker of nutrition and inflammation. It has recently emerged as a predictor of outcome after surgery for rectal cancer. Our aim was to evaluate if pre-operative serum albumin would predict survival after resection for rectal cancer.Method226 Patients with rectal cancer of all stages undergoing resection with curative intent were studied. Kaplan-Meier curves analysed survival based on a pre-operative albumin level of <35g/L vs. >35g/L. We sought for significant associations of survival with age, sex, stage, tumour site, use of neoadjuvant chemoradiation, microscopic positive resection margins, differentiation, angio, peri-neural, and lymphovascular invasion using individual variable analysis. Multifactorial analysis was performed using type III analysis with Weibull hazard model and Cox-proportional hazard model. Significance was assigned to a P value <0.05.
Results:
Of 226 patients (median age- 59 years; range 19 -- 88, Male - 54%), forty five (20%) had an albumin level < 35g/L and was associated with a poor overall survival (P = 0.02). Mean survival in months for <35g/L vs. >35g/L was 64.7 (SE - 9.3) vs. 95.8 (SE -- 7.0) and 5 year overall survival rates were 49% and 69%. Individual variable analysis revealed age, circumferential margin, stage, perineural, lympho-vascular and angio invasion to be also significant. With multifactorial analysis hypoalbuminaemia (HR = 0.58; 95% CI: 0.35 - 0.95, P = 0.03), advanced stage (HR = 2.0; 95% CI: 1.26 - 3.23, P < 0.01) and positive circumferential margin (HR = 2.2; 95% CI: 1.26 - 3.89, P < 0.01) remained significant.
Conclusion:
Preoperative hypoalbuminaemia is an independent risk factor for poor overall survival in rectal cancer. Advanced tumour stage and circumferential margin positivity were the other associations with poor survival.
Background:
The identification of serial miRNAs targeting the same functional gastric protein could provide new and effective serological biomarkers for the diagnosis of gastric cancer (GC). The aim of this study was to evaluate the potential of miR-20a-5p, let-7a and miR-320a in the diagnosis of AG or GC and the correlation of the three miRNAs with their predicted target molecules PGA, PGC and PGA/PGC ratio.
Methods:
The total of 291 patients included 103 controls (CON), 94 with atrophic gastritis (AG) and 94 with GC. The levels of serum miRNAs were detected by quantitative reverse transcription-polymerase chain reaction and serum pepsinogen A (PGA) and C (PGC) were determined by enzyme-linked immunosorbent assays.
Results:
Serum miR-320a level decreased through the controls, AG and GC groups which were the cascades of GC development, while there were no significant differences in levels of miR-20a-5p and let-7a among the controls, AG and GC groups. When stratified by gender and age, serum miR-320a expression was lower in female GC patients than in controls (p = 0.035), especially in female GC patients older than 60 years (p = 0.008). For distinguishing female GC patients aged over 60, the area under the receiver operating characteristic curve for miR-320a was 0.699, and the best cut-off point was 4.76 with a sensitivity of 65.2% and specificity of 68.2%. Concerning the correlations between the selected miR-20a-5p, let-7a, miR-320a and PGs, we found that there were positive correlations between all the three and the ratio of PGA/PGC (r = 0.408, 0.255, 0.324; p = <0.001, 0.009, 0.001, respectively), but there was no relationship between the expression of serum miR-20a-5p and its predicted target PGA, or between let-7a and miR-320a and their predicted target PGC. Serum miR-320a was decreased and PGC was increased in the GC group compared with the control group.
Conclusions:
Levels of serum miR-320a were lower in female GC patients older than 60 than in controls, which may provide a potential valuable marker for diagnosing older women with GC. The levels of serum miR-20a-5p, let-7a and miR-320a were positively correlated with PGA/PGC, which may indirectly reflect the functional status of the gastric mucosa.
Background:
Mucoepidermoid carcinoma (MEC) can be classified into low-, intermediate-, and high-grade tumors based on its histological features. MEC is mainly composed of three cell types (squamous or epidermoid, mucous and intermediate cells), which correlates with the histological grade and reflects its clinical behavior. Most cancers exhibit reduced methylation of repetitive sequences such as Long INterspersed Element-1 (LINE-1) and Alu elements. However, to date very little information is available on the LINE-1 and Alu methylation status in MEC. The aim of this study was to investigate LINE-1 and Alu element methylation in MEC and compare if key differences in the methylation status exist between the three different cell types, and adjacent normal salivary gland cells, to see if this may reflect the histological grade.
Methods:
LINE-1 and Alu element methylation of 24 MEC, and 14 normal salivary gland tissues were compared using Combine Bisulfite Restriction Analysis (COBRA). Furthermore, the three different cell types from MEC samples were isolated for enrichment by laser capture microdissection (LCM), essentially to see if COBRA was likely to increase the predictive value of LINE-1 and Alu element methylation.
Results:
LINE-1 and Alu element methylation levels were significantly different (p<0.001) between the cell types, and showed a stepwise decrease from the adjacent normal salivary gland to the intermediate, mucous and squamous cells. The reduced methylation levels of LINE-1 were correlated with a poorer histological grade. In addition, MEC tissue showed a significantly lower level of LINE-1 and Alu element methylation overall compared to normal salivary gland tissue (p<0.001).
Conclusions:
Our findings suggest that LINE-1 methylation differed among histological grade mucoepidermoid carcinoma. Hence, this epigenetic event may hold value for MEC diagnosis and prognostic prediction.
Background:
Population genetic studies focus on natural dispersal and isolation by landscape barriers as the main drivers of genetic population structure. However, anthropogenic factors such as reintroductions, translocations and wild x domestic hybridization may also have strong effects on genetic population structure. In this study we genotyped 351 Single Nucleotide Polymorphism markers evenly spread across the genome in 645 wild boar (Sus scrofa) from Northwest Europe to evaluate determinants of genetic population structure.
Results:
We show that wild boar genetic population structure is influenced by historical reintroductions and by genetic introgression from domestic pigs. Six genetically distinct and geographically coherent wild boar clusters were identified in the Netherlands and Western Germany. The Dutch Veluwe cluster is known to be reintroduced, and three adjacent Dutch and German clusters are suspected to be a result of reintroduction, based on clustering results, low levels of heterozygosity and relatively high genetic distances to nearby populations. Recent wild x domestic hybrids were found geographically widespread across clusters and at low frequencies (average 3.9%). The relationship between pairwise kinship coefficients and geographic distance showed male-biased dispersal at the population genetic level.
Conclusions:
Our results demonstrate that wildlife and landscape management by humans are shaping the genetic diversity of an iconic wildlife species. Historical reintroductions, translocation and recent restocking activities with farmed wild boar have all influenced wild boar genetic population structure. The current trend of wild boar population growth and range expansion has recently led to a number of contact zones between clusters, and further admixture between the different wild boar clusters is to be expected.
Background:
Calving efficiency can be described as the measure of a cow's ability to produce viable offspring within a specific period of time. This trait is crucial in beef cattle because calves are necessary both for the production of beef and for heifer replacements. Recently, the number of calves produced at 4 years of age (NCP4) has been used to evaluate the calving efficiency of Japanese Black cattle. To identify variants associated with calving efficiency in Japanese Black cattle, we conducted a genome-wide association study (GWAS) using 688 animals with extreme NCP4 values selected from 15,225 animals.
Results:
We identified genetic variants on bovine chromosome 12 (BTA12) that were associated with NCP4. The General Transcription Factor IIF, polypeptide 2 (GTF2F2), located in the 132 kbp-associated region, proved to be in strong linkage disequilibrium. We found 16 associated variants in the promoter and the 3' UTR regions. Consistent with this finding, transcripts of GTF2F2 derived from the haplotype (Q) with the increased number of calves were 1.33-fold more abundant than q-derived transcripts. Furthermore, luciferase assays revealed that the activity of the 3' UTR, a region that includes nine SNPs, was higher in constructs with the Q haplotype than in those with the q haplotype by approximately 1.35-fold. In contrast, the activity of the promoter region did not differ between haplotypes. The association was replicated in an independent sample of 827 animals that were randomly selected from the remainder of the cohort from the same farms used in the GWAS. In the replicated population, the frequency of the Q haplotype is 0.313, and this haplotype accounts for 2.69% of the total phenotypic variance. The effect of the Q to q haplotype substitution on NCP4 was 0.054 calves. These findings suggest that variants in the 3' UTR of GTF2F2 affect the level of GTF2F2 mRNA, which is associated with calving efficiency.
Conclusions:
This GWAS has identified variants in the 3' UTR of GTF2F2 that were associated with the NCP4 of Japanese Black cattle, and this association was validated in an independent sample. The Q haplotype will be immediately useful in improving the calving efficiency of Japanese Black cattle.
Background:
Mice homozygous for the juvenile alopecia mutation (jal) display patches of hair loss that appear as soon as hair develops in the neonatal period and persist throughout life. Although a report initially describing this mouse variant suggested that jal maps to mouse Chromosome 13, our preliminary mapping analysis did not support that claim.
Results:
To map jal to a particular mouse chromosome, we produced a 103-member intraspecific backcross panel that segregated for jal, and typed it for 93 PCR-scorable, microsatellite markers that are located throughout the mouse genome. Only markers from the centromeric tip of Chromosome 2 failed to segregate independently from jal, suggesting that jal resides in that region. To more precisely define jal's location, we characterized a second, 374-member backcross panel for the inheritance of five microsatellite markers from proximal Chromosome 2. This analysis restricted jal's position between D2Mit359 and D2Mit80, an interval that includes Il2ra (for interleukin 2 receptor, alpha chain), a gene that is known to be associated with alopecia areata in humans. Complementation testing with an engineered null allele of Il2ra, however, showed that jal is a mutation in a distinct gene. To further refine the location of jal, the 374-member panel was typed for a set of four single-nucleotide markers located between D2Mit359 and D2Mit80, identifying a 0.55 Mb interval where jal must lie. This span includes ten genes---only one of which, Gata3 (for GATA binding protein 3)---is known to be expressed in skin. Complementation testing between jal and a Gata3 null allele produced doubly heterozygous, phenotypically mutant offspring.
Conclusions:
The results presented indicate that the jal mutation is a mutant allele of the Gata3 gene on mouse Chromosome 2. We therefore recommend that the jal designation be changed to Gata3jal, and suggest that this mouse variant may provide an animal model for at least some forms of focal alopecia that have their primary defect in the hair follicle and lack an inflammatory component.
Background:
Since the first outbreak in Indonesia in 1926, Newcastle disease has become one of the most common and contagious bird diseases throughout the world. To date, enhancing host antibody response by vaccination remains the most efficient strategy to control outbreaks of Newcastle disease. Antibody response plays an important role in host resistance to Newcastle disease, and selection for antibody response can effectively improve disease resistance in chickens. However, the molecular basis of the variation in antibody response to Newcastle disease virus (NDV) is not clear. The aim of this study was to detect genes modulating antibody response to NDV by a genome-wide association study (GWAS) in chickens.
Results:
To identify genes or chromosomal regions associated with antibody response to NDV after immunization, a GWAS was performed using 39,833 SNP markers in a chicken F2 resource population derived from a cross between two broiler lines that differed in their resistance. Two SNP effects reached 5% Bonferroni genome-wide significance (P<1.26x10-6). These two SNPs, rs15354805 and rs15355555, were both on chicken (Gallus gallus) chromosome 1 and spanned approximately 600 Kb, from 100.4 Mb to 101.0 Mb. Rs15354805 is in intron 7 of the chicken Roundabout, axon guidance receptor, homolog 2 (ROBO2) gene, and rs15355555 is located about 243 Kb upstream of ROBO2. Rs15354805 explained 5% of the phenotypic variation in antibody response to NDV, post immunization, in chickens. Rs15355555 had a similar effect as rs15354805 because of its linkage disequilibrium with rs15354805 (r2=0.98).
Conclusion:
The region at about 100 Mb from the proximal end of chicken chromosome 1, including the ROBO1 and ROBO2 genes, has a strong effect on the antibody response to the NDV in chickens. This study paves the way for further research on the host immune response to NDV.
Background:
Whether or not a mutant allele in a population is under selection is an important issue in population genetics, and various neutrality tests have been invented so far to detect selection. However, detection of negative selection has been notoriously difficult, partly because negatively selected alleles are usually rare in the population and have little impact on either population dynamics or the shape of the gene genealogy. Recently, through studies of genetic disorders and genome-wide analyses, many structural variations were shown to occur recurrently in the population. Such "recurrent mutations" might be revealed as deleterious by exploiting the signal of negative selection in the gene genealogy enhanced by their recurrence.
Results:
Motivated by the above idea, we devised two new test statistics. One is the total number of mutants at a recurrently mutating locus among sampled sequences, which is tested conditionally on the number of forward mutations mapped on the sequence genealogy. The other is the size of the most common class of identical-by-descent mutants in the sample, again tested conditionally on the number of forward mutations mapped on the sequence genealogy. To examine the performance of these two tests, we simulated recurrently mutated loci each flanked by sites with neutral single nucleotide polymorphisms (SNPs), with no recombination. Using neutral recurrent mutations as null models, we attempted to detect deleterious recurrent mutations. Our analyses demonstrated high powers of our new tests under constant population size, as well as their moderate power to detect selection in expanding populations. We also devised a new maximum parsimony algorithm that, given the states of the sampled sequences at a recurrently mutating locus and an incompletely resolved genealogy, enumerates mutation histories with a minimum number of mutations while partially resolving genealogical relationships when necessary.
Conclusions:
With their considerably high powers to detect negative selection, our new neutrality tests may open new venues for dealing with the population genetics of recurrent mutations as well as help identifying some types of genetic disorders that may have escaped identification by currently existing methods.
Background:
Zebrafish embryos are transcriptionally silent until activation of the zygotic genome during the 10th cell cycle. Onset of transcription is followed by cellular and morphological changes involving cell speciation and gastrulation. Previous genome-wide surveys of transcriptional changes only assessed gene expression levels; however, recent studies have shown the necessity to map isoform-specific transcriptional changes. Here, we perform isoform discovery and quantification on transcriptome sequences from before and after zebrafish zygotic genome activation (ZGA).
Results:
We identify novel isoforms and isoform switches during ZGA for genes related to cell adhesion, pluripotency and DNA methylation. Isoform switching events include alternative splicing and changes in transcriptional start sites and in 3' untranslated regions. New isoforms are identified even for well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in cell-cell interactions such as f11r and magi1 display isoform switches with alterations of coding sequences. We also detect over 1000 transcripts that acquire a longer 3' terminal exon when transcribed by the zygote compared to their maternal transcript counterparts. ChIP-sequencing data mapped onto skipped exon events reveal a correlation between histone H3K36 trimethylation peaks and skipped exons, suggesting epigenetic marks being part of alternative splicing regulation.
Conclusions:
The novel isoforms and isoform switches reported here include regulators of transcriptional, cellular and morphological changes taking place around ZGA. Our data display an array of isoform-related functional changes and represent a valuable resource complementary to existing early embryo transcriptomes.
Background:
Primula species are important early spring garden plants with a centre of diversity and speciation in the East Himalaya-Hengduan Mountains in Western China. Studies on population genetics, speciation and phylogeny of Primula have been impeded by a lack of genomic resources. In the present study, we sequenced the transcriptomes of two closely related primrose species, Primula poissonii and Primula wilsonii, using short reads on the Illumina Genome Analyzer platform.
Results:
We obtained 55,284 and 55,011 contigs with N50 values of 938 and 1,085 for P. poissonii and P. wilsonii, respectively, and 6,654 pairs of putative orthologs were identified between the two species. Estimations of non-synonymous/synonymous substitution rate ratios for these orthologs indicated that 877 of the pairs may be under positive selection (Ka/Ks > 0.5), and functional enrichment analysis revealed that significant proportions of the orthologs were in the categories DNA repair, stress resistance, which may provide some hints as to how the two closely related Primula species adapted differentially to extreme environments, such as habitats characterized by aridity, high altitude and high levels of ionizing radiation. It was possible for the first time to estimate the divergence time between the radiated species pair, P. poissonii and P. wilsonii; this was found to be approximately 0.90 +/- 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Primers based on 54 pairs of orthologous SSR-containing sequences between the two Primula species were designed and verified. About half of these pairs successfully amplified for both species. Of the 959 single copy nuclear genes shared by four model plants (known as APVO genes), 111 single copy nuclear genes were verified as being present in both Primula species and exon-anchored and intron-spanned primers were designed for use.
Conclusion:
We characterized the transcriptomes for the two Primula species, and produced an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two Primula species not only revealed a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. Furthermore, we developed two sets of genetic markers, single copy nuclear genes and nuclear microsatellites (EST-SSR). Both these sets of markers will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.
Background:
RNA-seq has shown huge potential for phylogenomic inferences in non-model organisms. However, error, incompleteness, and redundant assembled transcripts for each gene in de novo assembly of short reads cause noise in analyses and a large amount of missing data in the aligned matrix. To address these problems, we compare de novo assemblies of paired end 90 bp RNA-seq reads using Oases, Trinity, Trans-ABySS and SOAPdenovo-Trans to transcripts from genome annotation of the model plant Ricinus communis. By doing so we evaluate strategies for optimizing total gene coverage and minimizing assembly chimeras and redundancy.
Results:
We found that the frequency and structure of chimeras vary dramatically among different software packages. The differences were largely due to the number of trans-self chimeras that contain repeats in the opposite direction. More than half of the total chimeras in Oases and Trinity were trans-self chimeras. Within each package, we found a trade-off between maximizing reference coverage and minimizing redundancy and chimera rate. In order to reduce redundancy, we investigated three methods: 1) using cap3 and CD-HIT-EST to combine highly similar transcripts, 2) only retaining the transcript with the highest read coverage, or removing the transcript with the lowest read coverage for each subcomponent in Trinity, and 3) filtering Oases single k-mer assemblies by number of transcripts per locus and relative transcript length, and then finding the transcript with the highest read coverage. We then utilized results from blastx against model protein sequences to effectively remove trans chimeras. After optimization, seven assembly strategies among all four packages successfully assembled 42.9--47.1% of reference genes to more than 200 bp, with a chimera rate of 0.92--2.21%, and on average 1.8--3.1 transcripts per reference gene assembled.
Conclusions:
With rapidly improving sequencing and assembly tools, our study provides a framework to benchmark and optimize performance before choosing tools or parameter combinations for analyzing short-read RNA-seq data. Our study demonstrates that choice of assembly package, k-mer sizes, post-assembly redundancy-reduction and chimera cleanup, and strand-specific RNA-seq library preparation and assembly dramatically improves gene coverage by non-redundant and non-chimeric transcripts that are optimized for downstream phylogenomic analyses.
Background:
Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated.
Results:
We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the 'single sample independence' (SSI) test that greatly reduced the number of false-positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the two metabolic states are associated with co-localization of additional transcription factors at CREB sites.
Conclusions:
Our results support a model in which CREB is constitutively bound to thousands of target genes, and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, and suggests a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx.
Background:
The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.
Results:
The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.
Conclusions:
This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.
Background:
Hepatitis C virus (HCV) is an RNA virus which has been known to cause acute and chronic necro-inflammatory disease of the liver. It is the leading cause of end-stage liver disease and hepatocellular carcinoma. HIV is known to have a negative impact on the natural disease outcome and immune response of HCV infection, whereas the reverse remains unclear. We evaluated the impact of HCV co-infection on recovery of CD4+ and CD8+ T-cells and liver enzyme levels before and after initiation of highly active antiretroviral therapy (HAART) in HIV/HCV co-infected patients.
Methods:
A hospital-based, observational, prospective cohort study design was used for this study. Pre-antiretroviral treatment (Pre-ART) and under HAART HIV mono-infected and HCV/HIV co-infected individuals who are under regular follow-up were recruited for this study. 387 blood samples were collected from volunteer, known HIV positive Ethiopian patients and screened for HCV. Twenty five HCV/HIV co-infected patients were prospectively followed for four years. CD4+ and CD8+ T-cells and liver enzyme levels were determined annually for each of the participant.
Results:
The prevalence of HCV/HIV co-infection in this study was 6.5%. Both HCV/HIV co-infected and HIV mono-infected under HAART groups showed CD4+ recovery (343 Vs 426; P < 0.004, OR = 4.97, 95% CI = 2.41 to 10.27) respectively; but, the recovery rate was higher in mono-infected (80 Vs 426) than co-infected group (148 Vs 343). The recovery and/or decline pattern of CD8+ T-cells was the same with that of CD4+. In 75% of co-infected groups, the mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were above the upper limit of normal reference range. Analyses restricted to individuals who initiated HAART and pre-ART showed similar results.
Conclusion:
We found that CD4+ T-cell recovery was negatively affected by the presence of ongoing HCV replication in under HAART co-infected individuals and fast decline of CD4+ T-cells in pre-ART patients. It was also associated with increased ALT and AST enzyme levels in both HAART initiated and treatment naive co-infected patients.
Background:
Infection with parasite protozoa is a long-term health issue in tropical and subtropical regions throughout the world. The Toll-like receptor (TLR) signaling pathway is one of the first-responding defense systems against Leishmania. The aim of this study was to investigate the expression of TLR2 and TLR9 in jejunum and colon and its correlation with CD11c, CD11b, and CD14 receptors used as markers for dendritic cells and macrophages.
Methods:
Twenty four dogs infected with Leishmania infantum were used in this study. Cytometry was carried out in lamina propria cells from jejunum and colon using markers for TLR2, TLR9, CD11b, CD11c and CD14.
Results:
Cellular inflammatory exudate was diffuse in the mucosa and submucosa, predominately comprising mononuclear cells: plasma cells, macrophages, and lymphocytes. Despite the parasite load, microscopy showed no erosion was evident in the epithelial mucosa layers. The colon harbored more parasites than the jejunum. Flow cytometry revealed higher frequency of TLR2+ and CD11c+ dendritic cells in the colon than in the jejunum. Conversely, TLR9-expressing cells were more frequent in jejunum. Moreover, frequency of macrophages (CD11b+ and CD14+) expressing simultaneity TLR9 were lower in the colon than in jejunum, while CD11c+ cells predominated in the colon. Despite of the negative ELISA serum results, IL-10 and TNF-alpha were higher in jejunum than colon of infected animals. However, IL-4 was higher in colon than jejunum of infected animals. A higher expression these cytokines were demonstrated in infected dogs compared to uninfected dogs.
Conclusions:
There was no correlation between clinical signs and pathological changes and immunological and parasitological findings in the gastrointestinal tract in canine visceral leishmaniasis. However, jejunum showed a lower parasite load with increased frequency and expression of CD11b, TLR9, CD14/CD11b/TLR9 receptors and IL-10 and TNF-alpha cytokines. Conversely, the colon showed a higher parasite load along with increased frequency and expression of TLR2, CD11c receptors, and IL-4 cytokine. Thus, Leishmania infantum is able to interfere in jejunum increased expression of TLR2, TLR9, CD11b, CD14, CD14/CD11b/TLR9 receptors, IL-10, and TNF-alpha; and in colon increased expression of CD11c, TLR2, TLR9, CD11b, CD14 e, CD14/CD11b/TLR9 receptors, IL-10, and TNF-alpha.
Genetic polymorphisms observed in various disease states associated with sensitivity or resistance to specific treatments have been a robust area of investigation for decades, with the potential to allow clinicians to make evidence-based decisions on the appropriate course of treatment. This study aimed to evaluate whether genetic polymorphisms of the signal transducer and activator of transcription 6 gene (STAT6) could be associated with a sustained virological response (SVR) among patients infected with hepatitis C virus genotypes 1 and 2 (HCV-1 and HCV-2) who were treated with peginterferon plus ribavirin (PEG-IFNalpha-RBV). We analyzed the associations between SVR to PEG-IFNalpha-RBV therapy and 4 single nucleotide polymorphisms (SNPs) in STAT6. This study included Taiwanese Chinese patients infected with either HCV-1 (n = 265) or HCV-2 (n = 195) in the presence or absence of an SVR. Among the STAT6 SNPs examined, the dosage effect of the A allele and allele frequency in rs1059513 were inversely correlated with SVR in patients infected with HCV-1 (P = 0.0179 and P = 0.0235, respectively). This effect was not observed in patients infected with HCV-2. The GG, GGG, and GGGC STAT6 haplotypes comprising 2, 3, and 4 SNPs (rs1059513, rs703817, rs324015, and rs3024974) were found to be associated with SVR, and their presence may increase the probability of a successful treatment outcome in patients infected with HCV-1 (P = 0.0273, 0.0352, and 0.0368, respectively). Moreover, a multivariate logistic regression model for predicting an SVR revealed that the presence of the GGGC haplotype carriers mutually affected the outcome of PEG-IFNalpha-RBV treatment. The presence of STAT6 SNPs and the association with SVR demonstrated that STAT6 polymorphisms might influence the therapeutic outcomes of patients infected with HCV-1 under standard-of-care (SOC) treatment.
Background:
IL-17, a Th17 cell-derived proinflammatory molecule, has been found to play an important role in the pathogenesis of autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). While IL-17 receptor (IL-17R) is expressed in many immune-related cells, microglia, and astrocytes, it is not known whether IL-17 exerts a direct effect on neural stem cells (NSCs) and oligodendrocytes, thus inducing inflammatory demyelination in the central nervous system.
Methods:
We first detected IL-17 receptor expression in NSCs with immunostaining and real time PCR. We then cultured NSCs with IL-17 and determined NSC proliferation by neurosphere formation capability and cell number count, differentiation by immunostaining neural specific markers, and apoptosis of NSCs by flow cytometry.
Results:
NSCs constitutively express IL-17R, and when the IL-17R signal pathway was activated by adding IL-17 to NSC culture medium, the number of NSCs was significantly reduced and their ability to form neurospheres was greatly diminished. IL-17 inhibited NSC proliferation, but did not induce cytotoxicity or apoptosis. IL-17 hampered the differentiation of NSCs into astrocytes and oligodendrocyte precursor cells (OPCs). The effects of IL-17 on NSCs can be partially blocked by p38 MAPK inhibitor.
Conclusions:
IL-17 blocks proliferation of NSCs, resulting in significantly reduced numbers of astrocytes and OPCs. Thus, in addition to its proinflammatory role in the immune system, IL-17 may also play a direct role in blocking remyelination and neural repair in the CNS.
Background:
Renal injury of anti-glomerular basement membrane (GBM) disease is defined by the linear deposition of IgG along GBM and rapidly progressive glomerulonephritis. To date, the distribution of anti-GBM IgG subclasses on renal tissue is still unclear. In the current study, we investigated the deposition of the four IgG subclasses using immunohistochemistry in the renal biopsy specimens from 46 patients with anti-GBM disease.
Results:
All four IgG subclasses can be detected within the GBM. Anti-GBM IgG3 was detected in all patients (100%), with 39 (84.8%) patients presenting with weak segmental staining and 7 (15.2%) patients with strong linear deposition. Anti-GBM IgG2 was detected in 22 (47.8%) patients, with 20 (90.9%) patients having weak segmental deposition and 2 (9.1%) patients presenting strong linear staining. Anti-GBM IgG1 and IgG4 were detected in 9 (19.6%) and 7 (15.2%) patients, respectively. IgG deposition along tubular basement membrane (TBM) was also detected in 31 (67.4%) patients. Among them, the IgG subclass distribution was similar to that of the deposition within the GBM: IgG1 6.5% (2/31), IgG2 45.2% (14/31), IgG3 100% (31/31) and IgG4 9.7% (3/31). We observed increased inflammatory cell infiltration into the interstitium in patients with increased anti-TBM IgG3 deposits (P=0.031).
Conclusions:
Anti-GBM IgG3 predominantly deposits along GBM and TBM on renal biopsy specimens from patients with anti-GBM disease, which may be involved in the development of renal injury of the disease.
Contributing reviewersThe editors of BMC Medical Imaging would like to thank all our reviewers who have contributed to the journal in Volume 12 (2012).
Background:
The American Society of Nuclear Cardiology and the Society of Nuclear Medicine conclude that incorporation of attenuation corrected (AC) images in myocardial perfusion scintigraphy (MPS) will improve diagnostic accuracy. The aim was to investigate the value of adding AC stress-only images for the decision whether a rest study is necessary or not.
Methods:
1,261 patients admitted to 99mTc MPS were studied. The stress studies were interpreted by two physicians who judged each study as "no rest study necessary" or "rest study necessary", by evaluating NC stress-only and NC + AC stress-only images. When there was disagreement between the two physicians, a third physician evaluated the studies. Thus, agreement between 2 out of 3 physicians was evaluated.
Results:
The physicians assessed 214 more NC + AC images than NC images as "no rest study necessary" (17% of the study population). The number of no-rest-study-required was significantly higher for NC + AC studies compared to NC studies (859 vs 645 cases (p < 0.0001). In the final report according to clinical routine, ischemia or infarction was reported in 23 patients, assessed as "no rest study necessary" (22 NC + AC cases; 8 NC cases), (no statistically significant difference). In 11 of these, the final report stated "suspected/possible ischemia or infarction in a small area".
Conclusions:
Adding AC stress-only images to NC stress-only images reduce the number of unnecessary rest studies substantially.
Background:
The purpose of this study was to evaluate the utility of a noninvasive ultrasound-based method, vibro-acoustography (VA), for thyroid imaging and determine the feasibility and challenges of VA in detecting nodules in thyroid.
Methods:
Our study included two parts. First, in an in vitro study, experiments were conducted on a number of excised thyroid specimens randomly taken from autopsy. Three types of images were acquired from most of the specimens: X-ray, B-mode ultrasound, and vibro-acoustography. The second and main part of the study includes results from performing VA and B-mode ultrasound imaging on 24 human subjects with thyroid nodules. The results were evaluated and compared qualitatively.
Results:
In vitro vibro-acoustography images displayed soft tissue structures, microcalcifications, cysts and nodules with high contrast and no speckle. In this group, all of US proven nodules and all of X-ray proven calcifications of thyroid tissues were detected by VA. In vivo results showed 100% of US proven calcifications and 91% of the US detected nodules were identified by VA, however, some artifacts were present in some cases.
Conclusions:
In vitro and in vivo VA images show promising results for delineating the detailed structure of the thyroid, finding nodules and in particular calcifications with greater clarity compare to US. Our findings suggest that, with further development, VA may be a suitable imaging modality for clinical thyroid imaging.
Background:
Carotid plaque echolucency as detected by Color Doppler ultrasonography (CDUS) has been used as a potential marker of plaque vulnerability. However, contrast-enhanced ultrasound (CEUS) has recently been shown to be a valuable method to evaluate the vulnerability and neovascularization within carotid atherosclerotic plaques. The aim of this study was to compare CEUS and CDUS in the assessment of plaque vulnerability using transcranial color Doppler (TCD) monitoring of microembolic signals (MES) as a reference technique.
Methods:
A total of 46 subjects with arterial stenosis (>= 50%) underwent a carotid duplex ultrasound, TCD monitoring of MES and CEUS (SonoVue doses of 2.0 mL) within a span of 3 days. The agreement between the CEUS, CDUS, and MES findings was assessed with a chi-square test. A p-value less than 0.05 was considered statistically significant.
Results:
Neovascularization was observed in 30 lesions (44.4%). The vascular risk factors for stroke were similar and there were no age or gender differences between the 2 groups. Using CEUS, MES were identified in 2 patients (12.5%) within class 1 (non-neovascularization) as opposed to 15 patients (50.0%) within class 2 (neovascularization) (p = 0.023). CDUS revealed no significant differences in the appearance of the MES between the 2 groups (hyperechoic and hypoechoic) (p = 0.237).
Conclusion:
This study provides preliminary evidence to suggest that intraplaque neovascularization detected by CEUS is associated with the presence of MESs, where as plaque echogenicity on traditional CDUS does not. These findings argue that CEUS may better identify high-risk plaques.
Background:
Circle contact lenses, also known as color contact lenses and big eye contact lenses, are a type of cosmetic contact lens. It is not generally known that a circle contact lens usually contains iron oxide and other metals, which means their use during magnetic resonance imaging (MRI) is a potential hazard.Case presentationWe present a rare case of incidental discovery of circle contact lenses by MRI and MRI images of circle lenses in vitro.
Conclusions:
Circle contact lenses usually contain iron oxide, which is a known source of susceptibility artifact on MRI. Not only radiologists and radiographers but also referring physicians should be familiar with the imaging findings and potential risk of scanning circle contact lenses by MRI.
Background:
In medical informatics, psychology, market research and many other fields, researchers often need to analyze and model ranking data. However, there is no statistical software that provides tools for the comprehensive analysis of ranking data. Here, we present pmr, an R package for analyzing and modeling ranking data with a bundle of tools. The pmr package enables descriptive statistics (mean rank, pairwise frequencies, and marginal matrix), Analytic Hierarchy Process models (with Saaty's and Koczkodaj's inconsistencies), probability models (Luce model, distance-based model, and rank-ordered logit model), and the visualization of ranking data with multidimensional preference analysis.
Results:
Examples of the use of package pmr are given using a real ranking dataset from medical informatics, in which 566 Hong Kong physicians ranked the top five incentives (1: competitive pressures; 2: increased savings; 3: government regulation; 4: improved efficiency; 5: improved quality care; 6: patient demand; 7: financial incentives) to the computerization of clinical practice. The mean rank showed that item 4 is the most preferred item and item 3 is the least preferred item, and significance difference was found between physicians' preferences with respect to their monthly income. A multidimensional preference analysis identified two dimensions that explain 42% of the total variance. The first can be interpreted as the overall preference of the seven items (labeled as "internal/external"), and the second dimension can be interpreted as their overall variance of (labeled as "push/pull factors"). Various statistical models were fitted, and the best were found to be weighted distance-based models with Spearman's footrule distance.
Conclusions:
In this paper, we presented the R package pmr, the first package for analyzing and modeling ranking data. The package provides insight to users through descriptive statistics of ranking data. Users can also visualize ranking data by applying a thought multidimensional preference analysis. Various probability models for ranking data are also included, allowing users to choose that which is most suitable to their specific situations.
Background:
The Pathobiology of Prediabetes in A Biracial Cohort study is a prospective evaluation of the transition from normal to impaired glucose regulation among African American and Caucasian adults with parental type 2 diabetes. This report describes recruitment strategies and relative yields for the 376 enrolled subjects.
Methods:
Recruitment occurred over 3.4 years, with clinical and metabolic assessments during 2.1-5.5 years of quarterly follow-up. The major recruitment sources were advertisements, community outreach, and clinical facilities. Advertisements included newspaper, television, radio, Internet, distributed brochures, utility bill inserts, and direct mailing. Community outreach included screening events during religious gatherings and health fairs, and referral by friends and families. The category of clinical facilities covered all subjects referred by health workers or recruited through area clinics and hospitals.
Results:
57.7% of participants were African American and 42.3% were Caucasian; the mean age (+/- SD) was 44.2 +/- 10.6 years, and ~70% were female. Advertisements yielded 52.4% of all participants, compared to 34.8% from community outreach and 12.8% from clinical facilities (P for trend < 0.0001). More Caucasians than African Americans cited advertising as the source of study information, whereas more African Americans than Caucasians cited community outreach. The accrual from clinical facilities was similar in both groups.
Conclusions:
Advertisements and community outreach were robust recruitment sources for assembling a diverse longitudinal diabetes offspring cohort, but each had differential yields in African Americans and Caucasians. Thus, a multifaceted approach comprising passive and active components is needed to recruit a multiracial clinical research population.
The quality of a consultation provided by a physician can have a profound impact on the quality of care and patient engagement in treatment decisions. When the COPD Assessment Test (CAT) was developed, one of its aims was to aid the communication between physician and patient about the impact of COPD. We developed a novel study design to assess this in a primary care consultation.Primary care physicians across five countries in Europe conducted videoed consultations with six standardised COPD patients (played by trained actors) which had patient-specific issues that the physician needed to identify through questioning. Half the physicians saw the patients with the completed CAT, and half without. Independent assessors scored the physicians on their ability to identify and address the patient-specific issues, review standard COPD aspects, their understanding of the case and their overall performance. This novel study design presented many challenges which needed to be addressed to achieve an acceptable level of robustness to assess the utility of the CAT. This paper discusses these challenges and the measures adopted to eliminate or minimise their impact on the study results.
Background To optimize the planning of blood donations but also to continue motivating the volunteers it is important to streamline the practical organization of the timing of donations. While donors are asked to return for donation after a suitable period, still a relevant proportion of blood donors is deferred from donation each year due to a too low hemoglobin level.Rejection of donation may demotivate the candidate donor and implies an inefficient planning of the donation process. Hence, it is important to predict the future hemoglobin level to improve the planning of donors' visits to the blood bank.Methods The development of the hemoglobin prediction rule is based on longitudinal (panel) data from blood donations collected by Sanquin (the only blood product collecting and supplying organization in the Netherlands). We explored and contrasted two popular statistical models, i.e. the transition (autoregressive) model and the mixed effects model as plausible models to account for the dependence among subsequent hemoglobin levels within a donor.Results The predictors of the future hemoglobin level are age, season, hemoglobin levels at the previous visits, and a binary variable indicating whether a donation was made at the previous visit. Based on cross-validation, the areas under the receiver operating characteristic curve (AUCs) for male donors are 0.83 and 0.81 for the transition model and the mixed effects model, respectively; for female donors we obtained AUC values of 0.73 and 0.72 for the transition model and the mixed effects model, respectively.Conclusion We showed that the transition models and the mixed effects models provide a much better prediction compared to a multiple linear regression model. In general, the transition model provides a somewhat better prediction than the mixed effects model, especially at high visit numbers. In addition, the transition model offers a better trade-off between sensitivity and specificity when varying the cut-off values for eligibility in predicted values. Hence transition models make the prediction of hemoglobin level more precise and may lead to less deferral from donation in the future.
Background:
Rater agreement is important in clinical research, and Cohen's Kappa is a widely used method for assessing inter-rater reliability; however, there are well documented statistical problems associated with the measure. In order to assess its utility, we evaluated it against Gwet's AC1 and compared the results.
Methods:
This study was carried out across 67 patients (56% males) aged 18 to 67, with a mean SD of 44.13 +/- 12.68 years. Nine raters (7 psychiatrists, a psychiatry resident and a social worker) participated as interviewers, either for the first or the second interviews, which were held 4 to 6 weeks apart. The interviews were held in order to establish a personality disorder (PD) diagnosis using DSM-IV criteria. Cohen's Kappa and Gwet's AC1 were used and the level of agreement between raters was assessed in terms of a simple categorical diagnosis (i.e., the presence or absence of a disorder). Data were also compared with a previous analysis in order to evaluate the effects of trait prevalence.
Results:
Gwet's AC1 was shown to have higher inter-rater reliability coefficients for all the PD criteria, ranging from .752 to 1.000, whereas Cohen's Kappa ranged from 0 to 1.00. Cohen's Kappa values were high and close to the percentage of agreement when the prevalence was high, whereas Gwet's AC1 values appeared not to change much with a change in prevalence, but remained close to the percentage of agreement. A Schizoid sample revealed a mean Cohen's Kappa of .726 and a Gwet's AC1of .853 , which fell within the different level of agreement according to criteria developed by Landis and Koch, and Altman and Fleiss.
Conclusions:
Based on the different formulae used to calculate the level of chance-corrected agreement, Gwet's AC1 was shown to provide a more stable inter-rater reliability coefficient than Cohen's Kappa. It was also found to be less affected by prevalence and marginal probability than that of Cohen's Kappa, and therefore should be considered for use with inter-rater reliability analysis.
A member of the NF-kappaB signaling pathway, PoAkirin1, was cloned from a full-length cDNA library of Japanese flounder (Paralichthys olivaceus). The full-length cDNA comprises a 5[prime]UTR of 202 bp, an open reading frame of 564 bp encoding a 187-amino-acid polypeptide and a 521-bp 3[prime]UTR with a poly (A) tail. The putative protein has a predicted molecular mass of 21 kDa and an isoelectric point (pI) of 9.22. Amino acid sequence alignments showed that PoAkirin1 was 99% identical to the Scophthalmus maximus Akirin protein (ADK27484). Yeast two-hybrid assays identified two proteins that interact with PoAkirin1: PoHEPN and PoC1q. The cDNA sequences of PoHEPN and PoC1q are 672 bp and 528 bp, respectively. Real-time quantitative reverse-transcriptase polymerase chain reaction analysis showed that bacteria could induce the expressions of PoAkirin1, PoHEPN and PoC1q. However, the responses of PoHEPN and PoC1q to the bacterial challenge were slower than that of PoAkirin1. To further study the function of PoAkirin1, recombinant PoAkirin1 and PoHEPN were expressed in Escherichia coli and would be used to verify the PoAkirin1-PoHEPN binding activity. These results identified two proteins that potentially interact with PoAkirin1 and that bacteria could induce their expression.
The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.
Contributing reviewersThe editors of BMC Molecular Biology would like to thank all of our reviewers who have contributed to the journal in Volume 12 (2012).
Background:
The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-OvationTM One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor(R) 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR).
Results:
Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient.
Conclusions:
A transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.
Background:
MicroRNAs (miRNAs) are a type of non-coding small RNA ~22 nucleotides in length that regulate the expression of protein coding genes at the post-transcriptional level. Glycolytic and oxidative myofibers, the two main types of skeletal muscles, play important roles in metabolic health as well as in meat quality and production in the pig industry. Previous expression profile studies of different skeletal muscle types have focused on these aspects of mRNA and proteins; nonetheless, an explanation of the miRNA transcriptome differences between these two distinct muscles types is long overdue.
Results:
Herein, we present a comprehensive analysis of miRNA expression profiling between the porcine longissimus doris muscle (LDM) and psoas major muscle (PMM) using a deep sequencing approach. We generated a total of 16.62 M (LDM) and 18.46 M (PMM) counts, which produced 15.22 M and 17.52 M mappable sequences, respectively, and identified 114 conserved miRNAs and 89 novel miRNA*s. Of 668 unique miRNAs, 349 (52.25%) were co-expressed, of which 173 showed significant differences (P < 0.01) between the two muscle types. Muscle-specific miR-1-3p showed high expression levels in both libraries (LDM, 32.01%; PMM, 20.15%), and miRNAs that potentially affect metabolic pathways (such as the miR-133 and -23) showed significant differences between the two libraries, indicating that the two skeletal muscle types shared mainly muscle-specific miRNAs but expressed at distinct levels according to their metabolic needs. In addition, an analysis of the Gene Ontology (GO) terms and KEGG pathway associated with the predicted target genes of the differentially expressed miRNAs revealed that the target protein coding genes of highly expressed miRNAs are mainly involved in skeletal muscle structural development, regeneration, cell cycle progression, and the regulation of cell motility.
Conclusion:
Our study indicates that miRNAs play essential roles in the phenotypic variations observed in different muscle fiber types.
Background:
The basal forebrain (BF) regulates cortical activity by the action of cholinergic projections to the cortex. At the same time, it also sends substantial GABAergic projections to both cortex and thalamus, whose functional role has received far less attention. We used deep brain stimulation (DBS) in the BF, which is thought to activate both types of projections, to investigate the impact of BF activation on V1 neural activity.
Results:
BF stimulation robustly increased V1 single and multi-unit activity, led to moderate decreases in orientation selectivity and a remarkable increase in contrast sensitivity as demonstrated by a reduced semi-saturation contrast. The spontaneous V1 local field potential often exhibited spectral peaks centered at 40 and 70Hz as well as reliably showed a broad gamma-band (30-90Hz) increase following BF stimulation, whereas effects in a low frequency band (1-10Hz) were less consistent. The broad gamma-band, rather than low frequency activity or spectral peaks was the best predictor of both the firing rate increase and contrast sensitivity increase of V1 unit activity.
Conclusions:
We conclude that BF activation has a strong influence on contrast sensitivity in V1. We suggest that, in addition to cholinergic modulation, the BF GABAergic projections play a crucial role in the impact of BF DBS on cortical activity.
Background:
The vesicular B0AT3 transporter (SLC6A17), one of the members of the SLC6 family, is a transporter for neutral amino acids and is exclusively expressed in brain. Here we provide a comprehensive expression profile of B0AT3 in mouse brain using in situ hybridization and immunohistochemistry.
Results:
We confirmed previous expression data from rat brain and used a novel custom made antibody to obtain detailed co-labelling with several cell type specific markers. B0AT3 was highly expressed in both inhibitory and excitatory neurons. The B0AT3 expression was highly overlapping with those of vesicular glutamate transporter 2 (VGLUT2) and vesicular glutamate transporter 1 (VGLUT1). We also show here that Slc6a17mRNA is up-regulated in animals subjected to short term food deprivation as well as animals treated with the serotonin reuptake inhibitor fluoxetine and the dopamine/noradrenaline reuptake inhibitor bupropion.
Conclusions:
This suggests that the B0AT3 transporter have a role in regulation of monoaminergic as well as glutamatergic synapses.
Background:
Gamma-synuclein is a member of the synuclein family of cytoplasmic, predominantly neuron-specific proteins. Despite numerous evidences for the importance of gamma-synuclein in the control of monoamine homeostasis, cytoskeleton reorganization and chaperone activity, its role in the regulation of cognitive behavior still remain unknown. Our previous study revealed that gamma-synuclein knockout mice are characterized by high habituation scores. Since a number of processes including spatial memory of the environment may affect habituation, in the present study we have carried out behavioral evaluation of spatial and working memory in gamma-synuclein knockout mice.
Results:
Inactivation of gamma-synuclein gene led to the improvement of working memory in mice as revealed by passive and active avoidance tests. At the same time behavioral tests, designed to assess spatial learning and memory (Morris water maze and Object location tests), showed no differences between gamma-synuclein knockouts and wild type mice.
Conclusions:
These findings indicate that young mice with targeted inactivation of gamma-synuclein gene have improved working memory, but not spatial learning and memory. Our results suggest that gamma-synuclein is directly involved in the regulation of cognitive functions.
Background:
While cortical representations of intrinsic hand muscles have been extensively studied in healthy individuals, little is known about the representation of proximal upper limb muscles. Improving our understanding of normal shoulder function is important, given that shoulder musculoskeletal disorders affect approximately 20% of the population and are suspected to involve changes in central motor representations. The purpose of the study is to describe the motor representation (motor evoked potentials (MEP) amplitude at the hotspot, map area, normalized map volume and center of gravity) of the infraspinatus muscle in healthy individuals, and to explore the potential influence of hand dominance on this representation (i.e. symmetry of the excitability and of the location of motor map between sides), as well as the effect of age and gender on motor excitability.
Results:
Fifteen healthy participants took part in this study. No significant asymmetry between sides was observed for motor excitability (p = 0.14), map area (p = 0.73) and normalized map volume (p = 0.34). Moreover, no side x intensity interaction was found (p = 0.54), indicating similar stimulus response properties. No difference between sides was found in the location of infraspinatus motor representation, either in the mediolateral or anteroposterior axis (p > 0.10). Neither age nor gender influenced aMT (p > 0.58) or MEP size (p > 0.61).
Conclusions:
As the cortical representation of infraspinatus muscles was found to be symmetric between sides, both in terms of excitability and location, comparisons between the intact and affected side could be performed in clinical studies, regardless of whether the dominant or non-dominant side is affected. The next step will be to characterize corticospinal excitability and map parameters in populations with shoulder disorders.
Background:
The human auditory cortex automatically encodes acoustic input from the environment and differentiates regular sound patterns from deviant ones in order to identify important, irregular events. The Mismatch Negativity (MMN) response is a neuronal marker for the detection of sounds that are unexpected, based on the encoded regularities. It is also elicited by violations of more complex regularities and musical expertise has been shown to have an effect on the processing of complex regularities. Using magnetoencephalography (MEG), we investigated the MMN response to salient or less salient deviants by varying the standard probability (70%, 50% and 35%) of a pattern oddball paradigm. To study the effects of musical expertise in the encoding of the patterns, we compared the responses of a group of non-musicians to those of musicians.
Results:
We observed significant MMN in all conditions, including the least salient condition (35% standards), in response to violations of the predominant tone pattern for both groups. The amplitude of MMN from the right hemisphere was influenced by the standard probability. This effect was modulated by long-term musical training: standard probability changes influenced MMN amplitude in the group of non-musicians only.
Conclusion:
This study indicates that pattern violations are detected automatically, even if they are of very low salience, both in non-musicians and musicians, with salience having a stronger impact on processing in the right hemisphere of non-musicians. Long-term musical training influences this encoding, in that non-musicians benefit to a greater extent from a good signal-to-noise ratio (i.e. high probability of the standard pattern), while musicians are less dependent on the salience of an acoustic environment.
Background:
Chlamydiaceae is a family of obligate intracellular pathogens with a worldwide distribution in many animal species, including humans. No information exists on the prevalence of Chlamydia felis infections in cats and dogs in Lanzhou, the geographical center of China. The aim of this study was to carry out a census of cats and dogs in Lanzhou and document the seroprevalence of C. felis exposure in these companion animals.
Results:
In this study, blood samples were collected from 485 animals (221 cats and 264 pet dogs) in Lanzhou between November 2010 and July 2011 to identify antibodies against C. felis. Thirteen of 221 (5.9%) cats and 32 of 264 (12.1%) pet dogs were positive for C. felis infection using indirect hemagglutination at a cutoff of 1:16. The seroprevalence in household and stray cats was 3.9% and 14.3%, respectively, and the difference was statistically significant (P < 0.05). Among different age groups, the seroprevalence in cats varied from 1.9 to 7.9%, and that in dogs ranged from 9.6 to 20.4%; however, the differences were not statistically significant (P > 0.05).
Conclusions:
This is the first report of the seroprevalence of C. felis exposure in cats and dogs in Lanzhou, northwestern China. Our results indicate that the presence of C. felis exposure in cats and dogs may pose a potential threat to human health.
Background:
Vibriosis caused by V. anguillarum is a commonly encountered disease in Atlantic cod farms and several studies indicate that the initiation of infection occurs after the attachment of the pathogen to the mucosal surfaces (gut, skin and gills) of fish. Therefore it is necessary to investigate the role of different mucosal components in fish upon V. anguillarum infection. The present study has two parts; in the first part we analyzed the differential expression of skin mucus proteins from Atlantic cod naturally infected with V. anguillarum using two dimensional gel electrophoresis coupled with mass spectrometry. In the second part, a separate bath challenge experiment with V. anguillarum was conducted to assess the mRNA levels of the genes in skin tissue, corresponding to the selected proteins identified in the first part.
Results:
Comparative proteome analysis of skin mucus of cod upon natural infection with V. anguillarum revealed key immune relevant proteins like calpain small subunit 1, glutathione-S-transferase omega 1, proteasome 26S subunit, 14-kDa apolipoprotein, beta 2-tubulin, cold inducible RNA binding protein, malate dehydrogenase 2 (mitochondrial) and type II keratin that exhibited significant differential expression. Additionally a number of protein spots which showed large variability amongst individual fish were also identified. Some of the proteins identified were mapped to the immunologically relevant JNK (c-Jun N-terminal kinases) signalling pathway that is connected to cellular events associated with pathogenesis. A bath challenge experiment with V. anguillarum showed differential expression of beta 2-tubulin, calpain small subunit 1, cold inducible RNA binding protein, flotillin1, and glutathione S-transferase omega 1 transcripts in the skin tissue of cod during early stages of infection.
Conclusions:
Differentially expressed proteins identified in the cod skin mucus point towards their possible involvement in V. anguillarum pathogenesis. The role of some of these proteins in vibriosis in cod described in this paper can be considered unconventional with respect to their established functions in higher vertebrates. Based on the differential expression of these proteins they are possibly important components of fish defence against bacteria and innate immunity at large. The feasibility of utilizing these proteins/genes as markers of bacterial infection or stress in cod needs to be explored further.
Background:
Little is known about ethnic, cultural and socioeconomic differences in childhood ownership and attitudes to pets. The objective of this study was to describe the factors associated with living with different pet types, as well as factors that may influence the intensity of relationship or 'attachment' that children have to their pet. Data were collected using a survey of 1021 9--10 year old primary school children in a deprived area of the city of Liverpool, UK.
Results:
Dogs were the most common pet owned, most common 'favourite' pet, and species most attached to. Twenty-seven percent of dog-owning children (10% of all children surveyed) reported living with a 'Bull Breed' dog (which includes Pit Bulls and Staffordshire Bull Terriers), and the most popular dog breed owned was the Staffordshire Bull Terrier. Multivariable regression modelling identified a number of variables associated with ownership of different pets and the strength of attachment to the child's favourite pet. Girls were more likely to own most pet types, but were no more or less attached to their favourite pet than boys. Children of white ethnicity were more likely to own dogs, rodents and 'other' pets but were no more or less attached to their pets than children of non-white ethnicity. Single and youngest children were no more or less likely to own pets than those with younger brothers and sisters, but they showed greater attachment to their pets. Children that owned dogs lived in more deprived areas than those without dogs, and deprivation increased with number of dogs owned. 'Pit Bull or cross' and 'Bull Breed' dogs were more likely to be found in more deprived areas than other dog types. Non-whites were also more likely to report owning a 'Pit Bull or cross' than Whites.
Conclusions:
Gender, ethnicity and socioeconomic status were associated with pet ownership, and sibling status with level of attachment to the pet. These are important to consider when conducting research into the health benefits and risks of the common childhood phenomenon of growing up with pets.
Background:
Oxidative stress, an excess of reactive oxygen species (ROS), causes lipid peroxidation resulting in cell and tissue damages. It may be associated with the development and progression of cancers in dogs. Malondialdehyde (MDA), the end product of lipid peroxidation, is commonly used as a marker of oxidative stress. The objective of this study was to assess oxidative stress in cancer-bearing dogs by measuring serum MDA levels. All client-owned dogs underwent physical examination at the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Khon Kaen University to determine the health status with the owner's consent. Blood samples of cancer-bearing dogs (N = 80) and clinically normal dogs (N = 101) were obtained and subjected for determination of MDA levels. In addition, complete blood count, creatinine, and alanine aminotransferase were measured.
Results:
Serum MDA was significantly higher in cancer-bearing dogs than in clinically normal dogs (mean +/- SD, 4.68 +/- 1.32 mumol/L vs 2.95 +/- 0.61 mumol/L, respectively; p < 0.001). Packed cell volume (mean +/- SD, 36.18 +/- 7.65% vs 44.84 +/- 5.54%), hemoglobin (mean +/- SD, 11.93 +/- 2.88 g% vs 15.17 +/- 2.00 g%) and red blood cells (median (IQA), 6.05 (2.15) vs 8.09 (1.34)) were all significantly lower in cancer-bearing dogs than in clinically normal dogs. However, the reverse was true for white blood cells (median (IQA), 18.20 (11.95) vs 14.90 (5.10)). Neither creatinine nor alanine aminotransferase levels were significantly different between groups.
Conclusions:
This study supports the conclusion that oxidative stress is associated with many types of cancers in dogs, as serum MDA levels were significantly higher in cancer-bearing dogs compared to clinically normal dogs.
Background:
Mycobacteria other than M. bovis may interfere with current bovine tuberculosis diagnostic tests resulting in false positive test results. As the prevalence of M. bovis decreases in the United States, interference from other mycobacteria play an increasingly important role in preventing the eradication of M. bovis. To identify mycobacteria other than M. bovis that may be interfering with current diagnostic tests, a retrospective study was performed to identify mycobacteria isolated from clinical tissues at the National Veterinary Services Laboratories between 1 January 2004 and 9 October 2011.
Results:
During the study period, 2,366 mycobacteria other than M. bovis were isolated from samples submitted for clinical diagnosis of M. bovis. Fifty-five mycobacterial species were isolated during this time period. In cattle, M. avium complex, M. fortuitum/fortuitum complex, M. smegmatis, M. kansasii, and M. terrae complex were the predominate species other than M. bovis isolated from tissues submitted for culture. Mycobacteria other than M. bovis isolated from deer were predominantly M. avium complex, M. terrae/terrae complex, and M. fortuitum/fortuitum complex.
Conclusions:
These data provide information characterizing the species and relative prevalence of mycobacteria other than M. bovis that may interfere with current diagnostic tests.
IntroductionOverdiagnosis in breast cancer screening is a controversial topic. One difficulty in estimation of overdiagnosis is the separation of overdiagnosis from lead time that is the advance in the time of diagnosis of cancers, which confers an artificial increase in incidence when a screening programme is introduced.
Methods:
We postulated a female population aged 50-79 with a similar age structure and age-specific breast cancer incidence as in England and Wales before the screening programme. We then imposed a two-yearly screening programme; screening women aged 50-69, to run for twenty years, with exponentially distributed lead time with an average of 40 months in screen-detected cancers. We imposed no effect of the screening on incidence other than lead time.
Results:
Comparison of age- and time-specific incidence between the screened and unscreened populations showed a major effect of lead time, which could only be adjusted for by follow-up for more than two decades and including ten years after the last screen. From lead time alone, twenty-year observation at ages 50-69 would confer an observed excess incidence of 37%. The excess would only fall below 10% with 25 years or more follow-up. For the excess to be nullified, we would require 30 year follow-up including observation up to 10 years above the upper age limit for screening.
Conclusions:
Studies using shorter observation periods will overestimate overdiagnosis by inclusion of cancers diagnosed early due to lead time among the nominally overdiagnosed tumours.
Breast cancer is a complex and heterogeneous disease that affects about one out of every eight women. In the last decade, several advancements have been made that have increased our understanding of breast cancer and have allowed us to more accurately diagnose and treat this disease in a more targeted manner. For example, gene expression profiling enabled the classification of breast cancers into four main subtypes - basal-like, HER2+ (human epidermal growth factor receptor 2-positive), luminal A and luminal B - and this classification is used to direct the use of targeted therapies such as tamoxifen or trastuzumab. The luminal subtypes are generally characterized as being estrogen receptor-positive and targetable with anti-hormone therapies. However, whereas luminal A cancers have a good prognosis, luminal B cancers are associated with early relapse following endocrine therapy and a prognosis that is similar to that of the aggressive basal subtype. It is thus imperative that luminal B cancers be better characterized so that therapeutic targets and biomarkers for this disease type can be realized. In the previous issue of Breast Cancer Research, Katchman and colleagues address this need by demonstrating that quiescin sulfydryl oxidase 1 (QSOX1), a secreted enzyme involved in post-translational modifications, is associated with poor prognosis in patients with luminal B breast cancer. The authors further determined that this protein promotes breast cancer proliferation and invasion. Collectively, these studies suggest that QSOX1 is a predictive biomarker for luminal cancers and that it may be a useful target for elusive luminal B disease.
IntroductionPercent mammographic density (PMD) adjusted for age and BMI is one of the strongest risk factors for breast cancer and is known to be approximately 60 percent heritable. Here we report a finding of an association between genetic ancestry and adjusted PMD.
Methods:
We selected self-identified Caucasian women in the California Pacific Medical Center Research Institute Cohort whose screening mammograms placed them in the top or bottom quintiles of age- and body mass index-adjusted PMD. Our final data set included 474 women with the highest adjusted PMD and 469 with the lowest genotyped on the Illumina 1M platform. Principal component analysis (PCA) and identity-by-descent (IBD) analyses allowed us to infer the women's genetic ancestry and correlate it with adjusted PMD.
Results:
Women of Ashkenazi Jewish ancestry, as defined by the first principal component (PC1) of PCA and identity-by-descent analyses, represented approximately 15 percent of the sample. Ashkenazi Jewish ancestry, defined by PC1, was associated with higher adjusted PMD (p = 0.004). Using multivariate regression to adjust for epidemiologic factors associated with PMD, including age at parity and use of postmenopausal hormone therapy, did not attenuate the association.
Conclusion:
Women of Ashkenazi Jewish ancestry based on genetic analysis are more likely to have high age- and BMI-adjusted PMD. Ashkenazi Jews may have a unique set of genetic variants or environmental risk factors that increase mammographic density.
IntroductionDysregulation of the insulin-like growth factor-1 receptor (IGF-1R)/ PI3K/Akt pathway was shown to correlate with breast cancer disease progression. Cancer stem cells (CSCs) are a subpopulation within cancer cells which participate in tumor initiation, radio/chemoresistance and metastasis. In breast cancer, breast CSCs (BCSCs) were identified as CD24-CD44+ cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Elucidation of the role of IGF-1R in breast cancer stem cells (BCSCs) is crucial to the design of breast cancer therapies targeting BCSCs.
Methods:
IGF-1R expression in BCSCs and non-CSCs sorted from xenografts of human primary breast cancers was examined by FACS, western blot analysis and immunoprecipitation (IP). The role of IGF-1R in BCSCs was assessed by IGF-1R blockade with chemical inhibitor and gene silencing. The involvement of PI3K/Akt/mTOR as downstream pathway was studied by their phosphorylation status upon IGF-1R inhibition and the effects of chemical inhibitors of these signaling molecules on BCSCs. We also studied 16 clinical specimens of breast cancer for the expression of phosphor-Akt in the BCSCs by FACS.
Results:
Expression of phosphorylated IGF-1R was greater in BCSCs than in non-BCSCs from xenografts of human breast cancer which were supported by western blot and IP experiments. The sorted IGF-1R expressing cells displayed features of cancer stem/progenitors such as mammosphere formation in vitro and tumorigenicity in vivo, both of which were suppressed by knockdown of IGF-1R. A specific inhibitor of the IGF-1R, PPP, suppressed the phospho-AktSer473 and preferentially decreased ALDH+ BCSC populations of human breast cancer cells. Furthermore, PPP inhibited the capacity of CD24-CD44+ BCSCs to undergo epithelial-mesenchymal transition process with downregulation of mesenchymal markers. Inhibitors of signal molecules downstream of IGR-1R including PI3K/Akt/mTOR also reduced the ALDH + population of breast cancer cells. Furthermore, the mTOR inhibitor, rapamycin, suppressed BCSCs in vitro and in vivo.
Conclusions:
Our data support the notion that IGF-1R is a marker of stemness, and IGF-1R and its downstream PI3K/Akt/mTOR pathway are attractive targets for therapy directed against breast cancer stem/progenitors.
IntroductionOf the nearly 1.4 million new cases of breast cancer diagnosed each year, a large proportion is characterized as hormone receptor negative, lacking estrogen receptors (ER) and/or progesterone receptors (PR). Patients with receptor-negative tumors do not respond to current steroid hormone based therapies and generally have significantly higher risk of recurrence and mortality compared to patients with tumors that are ER- and/or PR-positive. Previous in vitro studies had shown that the progesterone metabolites, 5-dihydroprogesterone (5P) and 3-dihydroprogesterone (3HP), respectively, exhibit pro-cancer and anti-cancer effects on receptor-negative human breast cell lines. Here in vivo studies were conducted to investigate the ability of 5P and 3HP to control initiation, growth and regression of ER/PR-negative human breast cell tumors.
Methods:
ER/PR-negative human breast cells (MDA-MB-231) were implanted into mammary fat pads of immunosuppressed mice and the effects of 5P and 3HP treatments on tumor initiation, growth, suppression/regression and histopathology were assessed in five separate experiments. Specific radioimmunoassays and gas chromatography-mass spectrometry were used to measure 5P, 3HP and progesterone in mouse serum and tumors.
Results:
Onset and growth of ER/PR-negative human breast cell tumors were significantly stimulated by 5P and inhibited by 3HP. When both hormones were applied simultaneously, the stimulatory effects of 5P were abrogated by the inhibitory effects of 3HP and vice versa. Treatment with 3HP subsequent to 5P-induced tumor initiation resulted in suppression of further tumorigenesis and regression of existing tumors. The levels of 5P in tumors, regardless of treatment, were about 10-fold higher than the levels of 3HP and the 5P:3HP ratios were about 5-fold higher than in serum, indicating significant changes in endogenous synthesis of these hormones in tumorous breast tissues.
Conclusions:
The studies showed that estrogen/progesterone insensitive breast tumors are sensitive to, and controlled by, the progesterone metabolites 5P and 3HP. Tumorigenesis of ER/PR-negative breast cells is significantly enhanced by 5P and suppressed by 3HP, the outcome depending upon the relative concentrations of these two hormones in the microenvironment in the breast regions. The findings show that the production of 5P greatly exceeds that of 3HP in ER/PR-negative tumors and that treatment with 3HP can effectively block tumorigenesis and regress existing tumors. The results provide the first hormonal theory to explain tumorigenesis of ER/PR-negative breast tissues and support the hypothesis that a high 3HP-to-5P concentration ratio in the microenvironment may foster normalcy in non-cancerous breast regions. The findings suggest new diagnostics based on the relative levels of these hormones and new approaches to prevention and treatment of breast cancers based on regulating the levels and action mechanisms of anti- and pro-cancer progesterone metabolites.
Background:
Cranial radiation therapy has been used for the treatment of primary and metastatic brain tumors. A prominent feature of brain injury induced by the radiation therapy is hippocampal dysfunction, characterized by a decline in memory. Cdk5 plays an important role in memory formation. Abnormal Cdk5 activity is associated with neuronal apoptosis induced by neurotoxic stimuli. However, the roles of Cdk5 in hippocampal apoptosis in response to X-ray irradiation have not been explored.
Methods:
The expression of Cdk5 activators, p35 and p25, in hippocampal neurons was tested in both in vivo animal and in vitro couture after X-ray irradiation.
Results:
After X-ray irradiation at 20 Gy and 30 Gy in rats, the number of hippocampal neuronal pyknosis was increased, but the number of hippocampal neuron was decreased, in the hippocampal CA1 region of rats. In these animals undergone with X-ray irradiation, the expression of p35 was significantly down-regulated, but it was up-regulated in p25. These opposite expressions were also shown in the primary cultured hippocampal neurons with 30 Gy irradiation. The apoptosis induced by X-ray irradiation were significantly prevented by the pretreatment of Cdk5 inhibitor, roscovitine, in both in vivo and in vitro settings.
Conclusions:
X-ray irradiation resulted in a hippocampal neuronal apoptosis through up-regulation of p25, the Cdk5 activator. Hyperactivity of Cdk5 was involved in the pathogenesis of X-ray irradiation-induced hippocampal neuronal apoptosis. Blockade of Cdk5 signal pathway effectively protected neurons from the irradiation-induced brain injury.
Background:
The multidrug resistance (MDR) 1 gene encodes a 170-kDa membrane transporter called P-glycoprotein, which plays an important role in protecting cells against lipophilic xenobiotics by the way of an ATP-dependent cellular efflux mechanism. Three polymorphisms of MDR1, 3435C > T located in exon 26, 1236C > T in exon 12 and 2677G > T/A in exon 21 were the most extensively studied and were identified functionally important and ethnically diverse mapping to the gene region. Considering the potential influence of altering MDR1 activity, it is plausible that MDR1 polymorphisms might play a role in the development of cancer. Although the effects of MDR1 polymorphisms on susceptibility to human cancer have been investigated in many studies, the results still remain conflicting.
Methods:
To resolve these conflicts, we performed a quantitative synthesis of the association between these three polymorphisms and cancer risk, including 52 studies (15789 cases and 20274 controls) for 3435C > T polymorphism, 10 studies (2101 cases and 2842 controls) for 1236C > T polymorphism and 18 studies (3585 cases and 4351 controls) for 2677G > T/A polymorphism.
Results:
The stratified analyses for 3435C > T polymorphism, individuals with T-allele in 3435C > T had significantly higher ALL risks (TT versus CC: OR =1.286, 95% CI =1.123-1.474); significantly elevated risks were observed among Caucasian populations (TT versus CC: OR =1.276, 95% CI =1.112-1.464). When restricting the analysis to the source of controls, we found that HB (hospital-based) genetic models had higher risks (TT versus CC: OR =1.307, 95% CI =1.046-1.632), as well as in PB (population-based) genetic models (TT versus CC: OR =1.294, 95% CI =1.079-1.55).The T/A-allele frequency of 2677G > T/A polymorphism was associated with higher risk of cancer (TT + TA + AA vs. GG: OR =1.348, 95% CI =1.031-1.762), significantly elevated risks were observed among Asian populations (TT + TA + AA vs. GG: OR =1.642, 95% CI =1.340-2.012), and elevated risks could be associated with PB models (TT + TA + AA vs. GG: OR =1.641, 95% CI =1.018-2.646).
Conclusions:
Our meta-analysis suggested that 3435C > T polymorphism and 2677G > T/A polymorphism were associated with cancer risk when all studies were pooled together, while 1236C > T polymorphism not.
Background:
Endometrial cancer (EC) is the most common gynecologic malignancy, but the molecular events involved in the development and progression of EC remain unclear. This study aimed to explore epigenetic modification of genes and miRNAs involved in EC development.
Methods:
Ishikawa and AN3CA cells were treated with 5'-Aza-2-deoxycytidine or histone deacetylase inhibitor. The expression of miRNAs and related genes were detected by PCR and Western blot. Promoter methylation was detected by bisulfite specific PCR sequencing. The proliferation, colony formation, cell cycle progression, migration and invasion of EC cells were evaluated by MTT, soft agar assay, flow cytometry, wound healing and invasion assay, respectively.
Results:
Aberrant expression of miRNAs including miR-200b, miR130a/b, miR-625 and miR-222 was associated with tumorigenesis and metastasis in endometrial cancer. Silencing of miR-130b induced E-cadherin expression, while ectopic expression of miR-130b and knockdown of DICER1 increased the expression of Vimentin, zeb2, N-cadherin, Twist and Snail in EC cells. Furthermore, 5'-Aza-2-deoxycytidine and Histone deacetylase (HDAC) inhibitor inhibited the proliferation, colony formation, migration and invasion of EC cells, accompanied by reduced MMP secretion.
Conclusions:
Our study provides the first description of epigenetic modification of epithelial mesenchymal transition associated genes and miRNAs in EC cells, which are extensively involved in the regulation of gene expression and subsequent accumulation of malignant features of EC cells.
Background:
MicroRNAs are small RNA molecules that negatively regulate gene expression by translational inhibition or mRNA cleavage. The discovery that abnormal expression of particular miRNAs contributes to human disease, including cancer, has spurred growing interest in analysing expression profiles of these molecules. Quantitative polymerase chain reaction is frequently used for quantification of miRNA expression due to its sensitivity and specificity. To minimize experimental error in this system an appropriate endogenous control gene must be chosen. An ideal endogenous control gene should be expressed at a constant level across all samples and its expression stability should be unaffected by the experimental procedure.
Results:
The expression and validation of candidate control genes (4.5S RNA(H) A, Y1, 4.5S RNA(H) B, snoRNA, U87 and U6) was examined in 21 rat cell lines to establish the most suitable endogenous control for miRNA analysis in a rat model of cancer. The stability of these genes was analysed using geNorm and NormFinder algorithms. U87 and snoRNA were identified as the most stable control genes, while Y1 was least stable.
Conclusion:
This study identified the control gene that is most suitable for normalizing the miRNA expression data in rat. That reference gene will be useful when miRNAs expression are analyzed in order to find new miRNA markers for endometrial cancer in rat.
Background:
We studied the role of caspase-2 in apoptosis induction by taxanes (paclitaxel, novel taxane SB-T-1216) in breast cancer cells using SK-BR-3 (nonfunctional p53, functional caspase-3) and MCF-7 (functional p53, nonfunctional caspase-3) cell lines.
Results:
Both taxanes induced apoptosis in SK-BR-3 as well as MCF-7 cells. Caspase-2 activity in SK-BR-3 cells increased approximately 15-fold within 48 h after the application of both taxanes at the death-inducing concentration (100 nM). In MCF-7 cells, caspase-2 activity increased approximately 11-fold within 60 h after the application of taxanes (300 nM). Caspase-2 activation was confirmed by decreasing levels of procaspase-2, increasing levels of cleaved caspase-2 and the cleavage of caspase-2 substrate golgin-160. The inhibition of caspase-2 expression using siRNA increased the number of surviving cells more than 2-fold in MCF-7 cells, and at least 4-fold in SK-BR-3 cells, 96 h after the application of death-inducing concentration of taxanes. The inhibition of caspase-2 expression also resulted in decreased cleavage of initiator caspases (caspase-8, caspase-9) as well as executioner caspases (caspase-3, caspase-7) in both cell lines after the application of taxanes. In control cells, caspase-2 seemed to be mainly localized in the nucleus. After the application of taxanes, it was released from the nucleus to the cytosol, due to the long-term disintegration of the nuclear envelope, in both cell lines. Taxane application led to some formation of PIDDosome complex in both cell lines within 24 h after the application. After taxane application, p21WAF1/CIP1 expression was only induced in MCF-7 cells with functional p53. However, taxane application did not result in a significant increase of PIDD expression in either SK-BR-3 or MCF-7 cells. The inhibition of RAIDD expression using siRNA did not affect the number of surviving SK-BR-3 and MCF-7 cells after taxane application at all.
Conclusion:
Caspase-2 is required, at least partially, for apoptosis induction by taxanes in tested breast cancer cells. We suggest that caspase-2 plays the role of an apical caspase in these cells. Caspase-2 seems to be activated via other mechanism than PIDDosome formation. It follows the release of caspase-2 from the nucleus to the cytosol.
The nucleus of the cell serves to maintain, regulate, and replicate the critical genetic information encoded by the genome. Genomic DNA is highly associated with proteins that enable simple nuclear structures such as nucleosomes to form higher-order organisation such as chromatin fibres. The temporal association of regulatory proteins with DNA creates a dynamic environment capable of quickly responding to cellular requirements and distress. The response is often mediated through alterations in the chromatin structure, resulting in changed accessibility of specific DNA sequences that are then recognized by specific proteins. Anti-cancer drugs that target cellular DNA have been used clinically for over four decades, but it is only recently that nuclease specific drugs have been developed to not only target the DNA but also other components of the nuclear structure and its regulation. In this review, we discuss some of the new drugs aimed at primary DNA sequences, DNA secondary structures, and associated proteins, keeping in mind that these agents are not only important from a clinical perspective but also as tools for understanding the nuclear environment in normal and cancer cells.
Increasing attention is focusing on chromosomal and genome structure in cancer research due to the fact that genomic instability plays a principal role in cancer initiation, progression and response to chemotherapeutic agents. The integrity of the genome (including structural, behavioral and functional aspects) of normal and cancer cells can be monitored with direct visualization by using a variety of cutting edge molecular cytogenetic technologies that are now available in the field of cancer research. Examples are presented in this review by grouping these methodologies into four categories visualizing different yet closely related major levels of genome structures. An integrated discussion is also presented on several ongoing projects involving the illustration of mitotic and meiotic chromatin loops; the identification of defective mitotic figures (DMF), a new type of chromosomal aberration capable of monitoring condensation defects in cancer; the establishment of a method that uses Non-Clonal Chromosomal Aberrations (NCCAs) as an index to monitor genomic instability; and the characterization of apoptosis related chromosomal fragmentations caused by drug treatments.
Microarrays have been applied to the determination of genome-wide expression patterns during the cell cycle of a number of different cells. Both eukaryotic and prokaryotic cells have been studied using whole-culture and selective synchronization methods. The published microarray data on yeast, mammalian, and bacterial cells have been uniformly interpreted as indicating that a large number of genes are expressed in a cell-cycle-dependent manner. These conclusions are reconsidered using explicit criteria for synchronization and precise criteria for identifying gene expression patterns during the cell cycle. The conclusions regarding cell-cycle-dependent gene expression based on microarray analysis are weakened by arguably problematic choices for synchronization methodology (e.g., whole-culture methods that do not synchronize cells) and questionable statistical rigor for identifying cell-cycle-dependent gene expression. Because of the uncertainties in synchrony methodology, as well as uncertainties in microarray analysis, one should be somewhat skeptical of claims that there are a large number of genes expressed in a cell-cycle-dependent manner.
Background:
Although chromosome missegregation during oocyte maturation (OM) is a significant contributor to human morbidity and mortality, very little is known about the causes and mechanisms of aneuploidy. Several investigators have proposed that temporal perturbations during OM predispose oocytes to aberrant chromosome segregation. One approach for testing this proposal is to temporarily inhibit the activity of protein proteolysis during OM. We used the reversible proteasome inhibitor MG-132 to transiently perturb the temporal sequence of events during OM and subsequently analyzed mouse metaphase II (MII) for cytogenetic abnormalities. The transient inhibition of proteasome activity by MG-132 resulted in elevated levels of oocytes containing extra chromatids and chromosomes.
Results:
The transient inhibition of proteasome-mediated proteolysis during OM by MG-132 resulted in dose-response delays during OM and elevated levels of aneuploid MII oocytes. Oocytes exposed in vitro to MG-132 exhibited greater delays during metaphase I (MI) as demonstrated by significantly (p < 0.01) higher levels of MI arrested oocytes and lower frequencies of premature sister chromatid separation in MII oocytes. Furthermore, the proportions of MII oocytes containing single chromatids and extra chromosomes significantly (p < 0.01) increased with MG-132 dosage.
Conclusions:
These data suggest that the MG-132-induced transient delay of proteasomal activity during mouse OM in vitro predisposed oocytes to abnormal chromosome segregation. Although these findings support a relationship between disturbed proteasomal activity and chromosome segregation, considerable additional data are needed to further investigate the roles of proteasome-mediated proteolysis and other potential molecular mechanisms on chromosome segregation during OM.
Background:
To investigate potential mechanisms for telomere capture the spatial arrangement of telomeres and chromosomes was examined in G1 (non-cycling) mitotic cells with diploid or triploid genomes. This was examined firstly by directly labelling the respective short arm (p) and long arm subtelomeres (q) with different fluorophores and probing cell preparations using a number of subtelomere probe pairs, those for chromosomes 1, 3, 4, 5, 6, 7, 9, 10, 12, 17, 18, and 20. In addition some interstitial probes (CEN15, PML and SNRPN on chromosome 15) and whole chromosome paint probes (e.g. WCP12) were jointly hybridised to investigate the co-localization of interphase chromosome domains and tethered subtelomeres. Cells were prepared by omitting exposure to colcemid and hypotonic treatments.
Results:
In these cells a specific interphase chromosome topology was detected. It was shown that the p and q telomeres of the each chromosome associate frequently (80% pairing) in an intrachromosomal manner, i.e. looped chromosomes with homologues usually widely spaced within the nucleus. This p-q tethering of the telomeres from the one chromosome was observed with large (chromosomes 3, 4, 5), medium sized (6, 7, 9, 10, 12), or small chromosomes (17, 18, 20). When triploid nuclei were probed there were three tetherings of p-q subtelomere signals representing the three widely separated looped chromosome homologues. The separate subtelomere pairings were shown to coincide with separate chromosome domains as defined by the WCP and interstitial probes. The 20% of apparently unpaired subtelomeric signals in diploid nuclei were partially documented to be pairings with the telomeres of other chromosomes.
Conclusions:
A topology for telomeres was detected where looped chromosome homologues were present at G1 interphase. These homologues were spatially arranged with respect to one-another independently of other chromosomes, i.e. there was no chromosome order on different sides of the cell nuclei and no segregation into haploid sets was detected. The normal function of this high frequency of intrachromosomal loops is unknown but a potential role is likely in the genesis of telomere captures whether of the intrachromosomal type or between non-homologues. This intrachromosomal tethering of telomeres cannot be related to telomeric or subtelomeric sequences since these are shared in varying degree with other chromosomes. In our view, these intrachromosomal telomeric tetherings with the resulting looped chromosomes arranged in a regular topology must be important to normal cell function since non-cycling cells in G1 are far from quiescent, are in fact metabolically active, and these cells represent the majority status since only a small proportion of cells are normally dividing.
Parkinson's disease (PD) coincides with a dramatic loss of dopaminergic neurons within the substantia nigra. A key player in the loss of dopaminergic neurons is oxidative stress. Dopamine (DA) metabolism itself is strongly linked to oxidative stress as its degradation generates reactive oxygen species (ROS) and DA oxidation can lead to endogenous neurotoxins whereas some DA derivatives show antioxidative effects. Therefore, DA metabolism is of special importance for neuronal redox-homeostasis and viability.In this review we highlight different aspects of dopamine metabolism in the context of PD and neurodegeneration. Since most reviews focus only on single aspects of the DA system, we will give a broader overview by looking at DA biosynthesis, sequestration, degradation and oxidation chemistry at the metabolic level, as well as at the transcriptional, translational and posttranslational regulation of all enzymes involved. This is followed by a short overview of cellular models currently used in PD research. Finally, we will address the topic from a medical point of view which directly aims to encounter PD.
A fundamental property of hematopoietic stem cells (HSCs) is the ability to self-renew. This is a complex process involving multiple signal transduction cascades which control the fine balance between self-renewal and differentiation through transcriptional networks. Key activators/regulators of self-renewal include chemokines, cytokines and morphogens which are expressed in the bone marrow niche, either in a paracrine or autocrine fashion, and modulate stem cell behaviour. Increasing evidence suggests that the downstream signaling pathways induced by these ligands converge at multiple levels providing a degree of redundancy in steady state hematopoiesis. Here we will focus on how these pathways cross-talk to regulate HSC self-renewal highlighting potential therapeutic windows which could be targeted to prevent leukemic stem cell self-renewal in myeloid malignancies.
We investigated the influence of altered gravity on key proteins of T cell activation during the MASER-12 ballistic suborbital rocket mission of the European Space Agency (ESA) and the Swedish Space Cooperation (SSC) at ESRANGE Space Center (Kiruna, Sweden). We quantified components of the T cell receptor, the membrane proximal signaling, MAPK-signaling, IL-2R, histone modifications and the cytoskeleton in non-activated and in ConA/CD28-activated primary human T lymphocytes. The hypergravity phase during the launch resulted in a downregulation of the IL-2 and CD3 receptor and reduction of tyrosine phosphorylation, p44/42-MAPK phosphorylation and histone H3 acetylation, whereas LAT phosphorylation was increased. Compared to the baseline situation at the point of entry into the microgravity phase, CD3 and IL-2 receptor expression at the surface of non-activated T cells were reduced after 6 min microgravity. Importantly, p44/42-MAPK-phosphorylation was also reduced after 6 min microgravity compared to the 1g ground controls, but also in direct comparison between the in-flight mug and the 1g group. In activated T cells, the reduced CD3 and IL-2 receptor expression at the baseline situation recovered significantly during in-flight 1g conditions, but not during microgravity conditions. Beta-tubulin increased significantly after onset of microgravity until the end of the microgravity phase, but not in the in-flight 1g condition. This study suggests that key proteins of T cell signal modules are not severely disturbed in microgravity. Instead, it can be supposed that the strong T cell inhibiting signal occurs downstream from membrane proximal signaling, such as at the transcriptional level as described recently. However, the MASER-12 experiment could identify signal molecules, which are sensitive to altered gravity, and indicates that gravity is obviously not only a requirement for transcriptional processes as described before, but also for specific phosphorylation / dephosphorylation of signal molecules and surface receptor dynamics.
Background:
The Hippo-YAP signaling pathway is altered and implicated as oncogenic in many human cancers. However, extracellular signals that regulate the mammalian Hippo pathway have remained elusive until very recently when it was shown that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) ligands including lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P). LPA inhibits Lats kinase activity in HEK293 cells, but the potential involvement of a protein phosphatase was not investigated. The extracellular regulators of YAP dephosphorylation (dpYAP) and nuclear translocation in epithelial ovarian cancer (EOC) are essentially unknown.
Results:
We showed here that LPA dose- and time-dependently induced dpYAP in human EOC cell lines OVCA433, OVCAR5, CAOV3, and Monty-1, accompanied by increased YAP nuclear translocation. YAP was involved in LPA-induced migration and invasion of EOC cells and LPA3 was a major LPA receptor mediating the migratory effect. We demonstrated that G13, but not or to a lesser extent G12, Gi or Gq, was necessary for LPA-induced dpYAP and its nuclear translocation and that RhoA-ROCK, but not RhoB, RhoC, Rac1, cdc42, PI3K, ERK, or AKT, were required for the LPA-dpYAP effect. In contrast to results in HEK293 cells, LPA did not inhibit Mst and Lats kinase in OVCA433 EOC cells. Instead, protein phosphatase 1A (PP1A) acted down-stream of RhoA in LPA-induction of dpYAP. In addition, we identified that amphiregulin (AREG), a down-stream target of YAP which activated EGF receptors (EGFR), mediated an LPA-stimulated and EGFR-dependent long-term (16 hr) cell migration. This process was transcription- and translation-dependent and was distinct from a transcription- and YAP-independent short-term (4 hr) cell migration. EOC tissues had reduced pYAP levels compared to normal and benign ovarian tissues, implying the involvement of dpYAP in EOC pathogenesis, as well as its potential marker and/or target values.
Conclusions:
A novel LPA-LPA3-G13-RhoA-ROCK-PP1A-dpYAP-AREG-EGFR signaling pathway was linked to LPA-induced migration of EOC cells. Reduced pYAP levels were demonstrated in human EOC tumors as compared to both normal ovarian tissues and benign gynecologic masses. Our findings support that YAP is a potential marker and target for developing novel therapeutic strategies against EOC.
Background:
PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation.
Results:
PAG-depletion enhanced Src kinase activity and augmented proximal T-cell receptor signaling; exactly the phenotype expected for loss of this negative regulator. Surprisingly, rather than becoming hyper-proliferative, PAG-suppressed T cells became unresponsive. This was mediated by a Fyn-dependent hyper-phosphorylation of the inhibitory receptor CTLA-4, which recruited the protein tyrosine phosphatase Shp-1 to lipid rafts. Co-suppression of CTLA-4 abrogates this inhibition and restores proliferation to T cells.
Conclusion:
We have identified a fail-safe mechanism as well as a novel contribution of CTLA-4 to setting the activation threshold in T cells.
Background:
Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle.
Results:
Here, we show that CHK2 only localizes to centrosomes during mitosis. Using wild-type and CHK2-/- HCT116 human colon cancer cells and human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs we show that several CHK2 antibodies are non-specific and cross-react with an unknown centrosomal protein(s) by immunofluorescence. To characterize the localization of CHK2, we generated cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with the centrosomes in a Polo-like kinase 1-dependent manner during mitosis, from early mitotic stages until cytokinesis.
Conclusion:
Our findings demonstrate that a subpopulation of CHK2 localizes at the centrosomes in mitotic cells but not in interphase. These results are consistent with previous reports supporting a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis.
Background:
The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined.
Results:
We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry.
Conclusions:
"RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells.AvailabilityRodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).
Background:
Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3.
Methods:
We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation.
Results:
Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation.
Conclusions:
During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.
Proteins of the BTB-kelch family are known to be involved in multiple biological processes such as migration, cytoskeleton arrangement, regulation of cell morphology, protein ubiquitination and gene expression. KBTBD8 is a new member of this family. The gene was found in a comparative transcriptome analysis of pluripotent stem cells and was therefore suggested to play a role in the regulation of pluripotency. Comparative analysis of the gene and protein sequences revealed a high conservation throughout evolution especially in the characteristic domains of BTB, BACK and kelch. We identified the Golgi apparatus as the subcellular localization of the KBTBD8 protein in non-dividing cells and could show that KBTBD8 co-localizes with alpha-tubulin on the spindle apparatus of mitotic cells suggesting a role in cell proliferation. In conclusion, KBTBD8 is a new member of the BTB-kelch superfamily that is located in the Golgi apparatus and translocates to the spindle apparatus during mitosis.
Background:
During mitosis most nucleolar proteins redistribute to other locales providing an opportunity to study the relationship between nucleolar protein localization and function. Dictyostelium is a model organism for the study of several fundamental biological processes and human diseases but only two nucleolar proteins have been studied during mitosis: NumA1 and Snf12. Both of them are linked to the cell cycle. To acquire a better understanding of nucleolar protein localization and dynamics in Dictyostelium we studied the nucleolar localization of two additional proteins during mitosis: Snf12-linked forkhead-associated kinase A (FhkA), which is involved in the cell cycle, and Ca2+-binding protein 4a (CBP4a), which is a binding partner of NumA1.
The generation of a uniquely 32P-end-labeled DNA fragment is essential for DNA-binding experiments such as DNase I footprinting and ethylation interference. We describe here a protocol for end-labeling a restriction fragment. For a plasmid DNA bearing a region containing the binding site of interest, cleaving with a single restriction endonuclease generates a 5' overhang containing a phosphate. This is generally necessary for both common forms of fragment end-labeling: phosphorylation with polynucleotide kinase and "filling in the end" with DNA polymerases (e.g., Klenow fragment). For the phosphorylation reaction, as described here, the phosphate is removed with calf intestinal phosphatase or bacterial alkaline phosphatase, and the resulting free 5'-OH is phosphorylated with polynucleotide kinase and [-32P]ATP. This generates a plasmid labeled at each end with -32P. The molar amount of plasmid DNA must be below the amount of ATP added to the reaction and the ATP must be of sufficiently high specific activity to generate a fragment labeled to the extent necessary for many DNA-binding experiments. To generate a uniquely end-labeled DNA fragment, the labeled plasmid is heat-treated to inactivate any remaining kinase and recleaved with a second endonuclease, releasing a short DNA fragment and a longer vector fragment. The DNA fragment is purified from the labeled vector on a 5%–8% native polyacrylamide gel. The preparation and labeling of DNA restriction fragments typically takes 1–2 d.
Inducing RNAi in Drosophila Cells by Transfection with dsRNA (2013-05-01T06:00:31-07:00) RNA Interference (RNAi)/siRNA, Drosophila, Analysis of Gene Expression, RNA, Analysis of Gene Expression in Cultured Cells, Topic Intro 82 Protocols 5438-5445
In Drosophila cells, RNA interference (RNAi) can be triggered by synthetic long double-stranded RNAs (dsRNAs). For many Drosophila cell lines and cell types, passive dsRNA uptake is inefficient. More complete silencing responses can often be obtained in Drosophila S2 cells using transfection, perhaps because higher levels of intracellular dsRNA are achieved. In this protocol, S2 cells are transfected with dsRNA using QIAGEN's Effectene reagent, which has proven to be reliable for many investigators. A plasmid DNA can also be included in the transfection mix to provide additional functionality. The plasmid DNA can encode, for example, a reporter of the activity of a pathway or specific transcription factor, or a marker that allows visualization of some cellular behavior or structure. It is also useful to include a plasmid that encodes a fluorescent protein simply to monitor transfection efficiency.
High-throughput sequencing (HTS) methods can provide short sequence reads for many millions of individual molecules in a sample, allowing the use of sequencing to measure the abundance of RNA molecules. To quantify the amount of a particular sequence in a sample of large RNAs (e.g., mRNAs), it is important to fragment the RNA into short pieces that can be ligated to oligonucleotides that allow polymerase chain reaction (PCR) amplification and sequencing. The most desired end structure of RNA for such ligation steps is a 5' phosphate and a 3' OH. Thus, enzymes that leave these groups after cleavage are of particular utility, avoiding the need to dephosphorylate the 3' end with phosphatases or phosphorylate the 5' end with kinase before proceeding. One such enzyme, RNase III, is widely available. Although it primarily cuts duplex RNA, this specificity is salt- and concentration-dependent, and many RNAs that lack strong extended duplexes are nonetheless susceptible to cleavage at many spots. RNA fragmentation by RNase III does not seem to grossly affect the distribution of RNA sequencing reads. Thus, it has become a standard method for creating nominally representative pools of transcriptome sequences with 5' phosphates and 3' OH for library construction. Three steps in preparing fragmented transcriptome RNA for sequencing library construction are described here: (1) fragmenting the RNA with RNase III to the extent that ~60–100-nucleotide fragments are created, (2) purifying the RNA from the RNase III reaction, and (3) analyzing the digestion products for their suitability in library production.
Microinjecting Holo-Aequorin into Dechorionated and Intact Zebrafish Embryos (2013-05-01T06:00:31-07:00) Cell Biology, general, Signal Transduction, Calcium Imaging, Imaging Development, In Vivo Imaging, In Vivo Imaging, general, Zebrafish, Developmental Biology, Collections, Calcium Techniques, PROT 072777
The injection of holo-aequorin into embryos at the one-cell stage, along with the use of a simple photomultiplier tube or luminescence imaging system, allows transient localized elevations of free cytosolic Ca2+ to be recorded and observed during the first 24 h of zebrafish development. The technique for loading dechorionated or intact one-cell stage zebrafish embryos with holo-aequorin is described here.
DNase I Footprinting (2013-05-01T06:00:31-07:00) Molecular Biology, general, DNA Protein Interactions, Transcriptional Regulation 2nd edition
DNase I footprinting has found a wide following for both identifying and characterizing DNA–protein interactions, particularly because of its simplicity. The concept is that a partial digestion by DNase I of a uniquely 32P-end-labeled fragment will generate a ladder of fragments, whose mobilities on a denaturing acrylamide gel and whose positions in a subsequent autoradiograph will represent the distance from the end label to the points of cleavage. Bound protein prevents binding of DNase I in and around its binding site and thus generates a "footprint" in the cleavage ladder. The distance from the end label to the edges of the footprint represents the position of the protein-binding site on the DNA fragment. The position of the binding site can be determined by electrophoresing a DNA sequencing ladder alongside the footprint. DNase I cannot bind directly adjacent to a DNA-bound protein because of steric hindrance. Hence, the footprint gives a broad indication of the binding site, generally 8–10 base pairs (bp) larger than the site itself.
Background:
Alcoholic liver disease (ALD) is a significant cause of death and morbidity. Detection of liver fibrosis at an early stage could provide opportunities for more optimal management. Serum markers of liver fibrosis offer an alternative to biopsy. Evidence of the performance of biomarkers in ALD is needed and a systematic review to evaluate available studies was conducted.
Methods:
Electronic databases were searched. Studies were included if they evaluated paired samples of biopsy and serum, and presented data as sensitivity, specificity, or ROC curves.
Results:
15 studies were included- median participant number = 146 (range 44--1034). Studies differed with respect to patient populations. 6 single markers were evaluated (mostly Hyaluronic Acid), and ten combined panels. Biomarkers could discriminate between people with severe fibrosis/cirrhosis with high diagnostic accuracy- HA (median AUROC 0.79 range 0.69-0.93), panels (median AUROC 0.83 range 0.38-0.95). Significant heterogeneity precluded pooling. Performance was poorer for detecting less severe fibrosis.
Conclusions:
There are limited numbers of small studies evaluating the accuracy of biomarkers in identifying fibrosis on biopsy in ALD. Some showed promise (both HA alone and some panels) in the identification of cirrhosis/severe fibrosis and could be used to rule it out in heavy drinkers. Biomarkers less accurate with less severe fibrosis.
Background:
Liver cancer is one of the most common malignancies in the world and at the moment, there is no drug intervention effective for the treatment of liver tumours. Investigate the effect of N-acetylcysteine (NAC), which has been studied for its antitumoural properties, on the toxicity of hepatocarcinoma (HCC) cells in vitro when used with the drug interferon alpha-2A (IFN), which is used clinically to treat HCC.
Results:
NAC, IFN and NAC plus IFN reduced cell viability, as determined by MTT assay. More importantly, NAC potentiates the cytotoxic effect of IFN, with the best response achieved with 10 mM of NAC and 2.5 x 104 of IFN. These results were confirmed by Annexin/PI staining through flow cytometry and morphologic analyses. Co-treatment reduced the expression of the nuclear transcription factor kappa-B (NF-kB). In a similar way to NAC, RNAi against p65 potentiated the toxic effect of IFN, suggesting that, indeed, NAC may be enhancing the effect of IFN through inhibition of NF-kB.
Conclusions:
Our results support the notion that NAC may be an important drug for the treatment of liver tumours as primary or adjuvant therapy. IFN has a limited clinical response, and therefore, the anti-proliferative properties of NAC in the liver should be explored further as an alternative for non-responders to IFN treatment.
Background:
After partial hepatectomy (PHx), the liver regeneration process terminates when the normal liver-mass/body-weight ratio of 2.5% has been re-established. To investigate the genetic regulation of the terminating phase of liver regeneration, we performed a 60% PHx in a porcine model. Liver biopsies were taken at the time of resection, after three weeks and upon termination the sixth week. Gene expression profiles were obtained using porcine oligonucleotide microarrays. Our study reveals the interactions between genes regulating the cell cycle, apoptosis and angiogenesis, and the role of Transforming Growth Factor-beta (TGF-beta) signalling towards the end of liver regeneration.
Results:
Microarray analysis revealed a dominance of genes regulating apoptosis towards the end of regeneration. Caspase Recruitment Domain-Containing Protein 11 (CARD11) was up-regulated six weeks after PHx, suggesting the involvement of the caspase system at this time. Zinc Finger Protein (ZNF490) gene, with a potential negative effect on cell cycle progression, was only up-regulated at three and six weeks after PHx indicating a central role at this time. TGF-beta regulation was not found to be significantly affected in the terminating phase of liver regeneration. Vasohibin 2 (VASH2) was down-regulated towards the end of regeneration, and may indicate a role in preventing a continued vascularization process.
Conclusions:
CARD11, ZNF490 and VASH2 are differentially expressed in the termination phase of liver regeneration. The lack of TGF-beta up-regulation suggests that signalling by TGF-beta is not required for termination of liver regeneration.
Background:
This report presents a detailed description of hepatic architecture in 46 amphibian livers by light microscopy, and extensively discusses the phylogenetic viewpoint.
Results:
The 46 amphibian livers showed a variety of histological features, but anurans were the same as in mammalian livers. The hepatocyte-sinusoidal structures of the amphibian livers were classified into three different types: (I) several-cell-thick plate type, (II) two-cell-thick plate type, and (III) one-cell-thick plate type, depending on the percentage extension of sinusoidal areas per unit area, measured by morphometry. Hematopoietic tissue structures were observed in the connective tissue of both the perihepatic subcapsular regions and portal triads in the order Caudata and Gymnophiona, but were not observed in the order Anura (except for the genus Bombina and Xenopus). As phylogenetic relationships are branched from urodeles to anurans, the parenchyma arrangement progressed from the combined several- and two-cell-thick plate type to one-cell-thick plate type as seen in the mammalian liver type. In contrast, hematopoietic tissue structures were exactly the opposite and did not involve anurans.
Conclusions:
This study is the first to investigate amphibian livers phylogenically, and their architectural differences are shown in the route of hepatic ontogenesis. In this process, parenchymal arrangement formation is acquired phylogenically. The occurrence of hematopoietic cells may be related with the development of the systemic immune system in the spleen and bone marrow.
Background:
Type-2 Diabetes is a major health concern in the United States and other Westernized countries, with prevalence increasing yearly. There is a need to better model and predict adverse drug reactions, drug-induced liver injury, and drug efficacy in this population. Because transporters significantly contribute to drug clearance and disposition, it is highly significant to determine whether a severe diabetes phenotype alters drug transporter expression, and whether diabetic mouse models have altered disposition of acetaminophen (APAP) metabolites.
Results:
Transporter mRNA and protein expression were quantified in livers and kidneys of adult C57BKS and db/db mice, which have a severe diabetes phenotype due to a lack of a functional leptin receptor. The urinary excretion of acetaminophen-glucuronide, a substrate for multidrug resistance-associated proteins transporters was also determined. The mRNA expression of major uptake transporters, such as organic anion transporting polypeptide Slco1a1 in liver and kidney, 1a4 in liver, and Slc22a7 in kidney was decreased in db/db mice. In contrast, Abcc3 and 4 mRNA and protein expression was more than 2 fold higher in db/db male mouse livers as compared to C57BKS controls. Urine levels of APAP-glucuronide, -sulfate, and N-acetyl cysteine metabolites were higher in db/db mice.
Conclusion:
A severe diabetes phenotype/presentation significantly altered drug transporter expression in liver and kidney, which corresponded with urinary APAP metabolite levels.
Background:
Melanoma is a deadly disease affecting people worldwide. Genetic studies have identified different melanoma subtypes characterized by specific recurrently mutated genes and led tothe successful clinical introduction of targeted therapies. Hotspot mutations in SF3B1 mutations were recently reported in uveal melanoma. Our aim was to see if these mutationsalso occur in cutaneous melanoma.FindingsWe analyzed a cohort of 85 cutaneous melanoma including 22 superficial spreading, 24 acral-lentiginous, 36 nodular, and 3 lentigo-maligna melanomas. Exon 14 of SF3B1, containing thesite of recurrent mutations described in uveal melanoma, was sequenced in all samples. Additionally, NRAS exon 1 and 2 and BRAF exon 15 were sequenced in all, KIT exons 9, 11,13, 17, and 18 in 30 samples. High numbers of BRAFand NRAS mutations were identified with frequencies varying according to melanoma subtype. None of the samples were found to harbor a SF3B1 mutation.
Conclusions:
We conclude that recurrent mutations in codon 625 ofSF3B1 as reported in uveal melanoma are not present in most types of cutaneous melanoma. This highlightsthe genetic differences between cutaneous and uveal melanoma and the need for subtype specific therapeuticapproaches.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/5324677809600649
Placental site nodules (PSNs) and epithelioid trophoblastic tumors (ETTs) respectively represent non-neoplastic and neoplastic lesions of chorionic-type intermediate trophoblasts(ITs). Many patients with a PSN have a history of a cesarean section (CS) or therapeutic abortion. Recent evidence shows that a PSN may progress to an ETT. Herein, we describe acoexisting ETT and placental site trophoblastic tumor (PSTT) intimately associated with PSNs in the post-cesarean lower uterine segment of a 41-year-old woman. The patientpresented with abnormal vaginal bleeding 1 year after a cesarean delivery for her most recent pregnancy. We speculated that the neoplasms had transformed from PSNs, the formation of which was related to faulty expulsion of the placental tissue or abnormal colonization ofchorionic-type ITs during the CS. Neoplastic trophoblastic cells derived from PSNs displayed differentiation plasticity toward chorionic-type ITs and implantation site ITs that wererespectively constituted of an ETT and PSTT.Virtual slidesThe virtual slides for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1597949195882123
Canine Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are uncommonly reported in the ulnar, since they are underestimated relative to the more common spindle cell tumours of soft tissue. In dogs, MPNST accounts for 27% of nervous system tumours . In man, MPNST represents 5-10% of all soft tissue sarcomas and is often associated with neurofibromatosis type 1 (NF-1).An 8-year-old, 9 kg, female mixed-breed dog with a subcutaneous mass on theupper right side of the ulnar region was presented to the small animal research and teaching hospital of Tehran University. The dog was anorexic with general weakness. The mass (7 x 4 cm) was removed surgically and processed routinely. Microscopically, the mass was composed of highly cellular areas with a homogeneous population of round or spindle cells, high cellular pleomorphism, high mitotic index and various morphologic patterns. Furthermore, spindle cells arranged in densely or loosely sweeping fascicles, interlacingwhorls, or storiform patterns together with wavy cytoplasm, nuclear palisades, and round cells were arranged in sheets or cords with a meshwork of intratumoral nerve fibers. Inaddition, in this case the presence of neoplastic cells within the blood vessels was observed. Immunohistochemically, tumor was positive for vimentin and S-100 protein. Thehistopathologic features coupled with the S-100 and vimentin immunoreactivity led to a diagnosis of malignant neurofibroma.To the best of our knowledge, primary ulnar MPNST has not been reported in animals. This is the first documentation of an ulnar malignant peripheral nerve sheath tumour in a dog.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1310907815984587
Background:
It has been accepted that reversed halo sign (RHS) appeared on a computed tomography (CT) image in immunocompromised patients indicates an invasive fungal infection, but its pathophysiology remains obscure as to what this image implies. Therefore, the present reportdescribes detailed radiological and histopathological findings of a case of invasive pulmonary mucormycosis (IPM) presenting RHS with comparison to those from a lesion of discrete nodule caused by invasive pulmonary aspergillosis (IPA), and discusses the pathophysiological implications of this characteristic image.Case presentationRHS had been clinically noted at the time of recovering of bone marrow function of a 64-year-old Japanese man who had chemotherapy for his acute lymphoblastic leukemia.Histological examination of the surgically removed lung revealed a lesion of IPM. This was composed of coagulation necrosis of septa at the center of lesion with preservation of aircontent which was encompassed outer rim comprising triplet structure; liquefaction, consolidation, and organization from the inner to the outer layer. In addition, Micro-CTexamination confirmed reticular structure and monotonous high density at the central coagulation necrosis preserving air content and surrounding consolidation, and organizationlesion of the IPM lesion.
Conclusion:
Our investigations suggest that RHS might be understood as a kind of immune reconstitution syndrome and be the initial and prior status of air crescent sign.Virtual SlidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/3480054198968132
Background:
Sudden cardiac death resulting from acute myocardial infarction (AMI) constitutes a significant percentage of the caseload for forensic and clinical pathologists. When sudden death occurs at an early stage (<6 h), pathologists experience difficulty in the postmortem diagnosis of AMI. Because of the specific tissue distribution of S100A1 and its relationship with acute ischemic heart disease, this study aimed to evaluate the performance of S100A1 in the postmortem diagnosis of AMI.
Methods:
We constructed a rat model of AMI through permanent ligation of the left anterior descending coronary artery (LAD) to investigate the depletion of S100A1 from ischemic cardiomyocytes by immunohistochemistry and measuring S100A1 plasma concentrations by enzyme-linked immunosorbent assay at varying post-infarction intervals. In addition, immunohistochemical staining of S100A1 for definite infarction, suspected early infarction, and in normal human hearts, was also performed to test its practical feasibility for postmortem diagnosis of AMI at an early stage.
Results:
As early as 15 min after ligation of the LAD, depletion of S100A1 was observed in ischemic cardiomyocytes, and S100A1 plasma concentration was also significantly higher than that of the sham-operated group (P < 0.001). With continuation of the occlusion time, the depleted areas of S100A1 further expanded and S100A1 plasma concentrations further increased. For autopsy material, all human cases of definite myocardial infarction and suspected early infarction showed well-defined areas without S100A1 staining. None of the normal human cases showed diffuse depletion of S100A1.
Conclusion:
Our results suggest that immunohistochemical detection of S100A1 is useful for the postmortem diagnosis of AMI at an early stage.
Background:
Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely-packed protuberances on its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan.ResearchThe genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101x and 5.2x. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has sixteen COG2132 multi-copper oxidase family proteins with root specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment.
Conclusions:
The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.
Background:
Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma and multiple myeloma.
Results:
Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (>100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways.
Conclusion:
This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network.
Background:
Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a CRISPR locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct Cas proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers' targets.
Results:
We discover 95,000 contigs that are putative invasive mobile genetic elements (MGEs), some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of MGEs than stool samples. MGEs carry genes encoding diverse functions: only 7% of the MGEs are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large MGEs of various types, and a relative abundance analysis of MGEs and putative hosts, exploring the dynamic activities of MGEs in human microbiomes. A joint analysis of MGEs and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target MGEs lacking motifs.
Conclusions:
We identify a large collection of invasive MGEs in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders.
Adenosquamous carcinoma (ASC) is a rare malignant tumor in the head and neck region and usually it occurs as a mucosal carcinoma. The World Health Organization classification1 shows this tumor as a variation of squamous cell carcinoma (SCC), whereas the Armed Forces Institute of Pathology (AFIP) classification2 categorized it as a salivary gland tumor. In the AFIP guidelines, the occurrence of ASC is limited to the minor salivary glands not in the major salivary glands.
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Esophageal hyperkeratosis is rarely described. In contrast to hyperkeratosis of orolaryngeal mucosa – where its risk factors and association with squamous neoplasia are well-studied – the prevalence and clinicopathologic features of esophageal hyperkeratosis are unknown.
Methods and results
We prospectively reviewed 1,845 esophageal biopsies and found hyperkeratosis in 37 (2.0%). Among 98 patients studied, hyperkeratosis occurred in two distinct settings: Group 1 (within Barrett's esophagus [BE]/adenocarcinoma, n=61, 62%), and Group 2 (outside BE/adenocarcinoma, n=37, 38%). In contrast to Group 1, hyperkeratosis in Group 2 was more often multifocal (>3 foci in 51% vs. 16%, P=0.0001), involved mid esophagus (51% vs. 2%, P<0.0001), showed endoscopic leukoplakia (24% vs. 3%, P=0.003), and involved current/former alcohol users (51% vs. 19%, P=0.0012). Importantly, invasive squamous carcinoma and squamous dysplasia were seen only in Group 2 (47% and 19% vs. 0%, P<0.0001). Further, 42% of Group 2, but none of Group 1, had benign or malignant squamous lesions of the oral cavity/larynx (P<0.0001).
Conclusion
Hyperkeratosis involves ~2% of esophageal biopsies and can be divided into cases occurring within BE/adenocarcinoma and those occurring outside BE/adenocarcinoma. The former lack clinical significance, whereas the latter are frequently associated with esophageal squamous neoplasia and squamous pathology of the head&neck region.
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Mucinous adenocarcinoma (MUC) is a commonly studied histological subtype of colorectal adenocarcinoma. However, the prognostic value of MUC remains unclear, particularly in patients stratified by the primary tumor site. We aimed to analyze the prognostic value of MUC in colorectal cancer.
Methods and Results
We utilized two independent datasets in this study: 1) the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) dataset; and 2) the dataset from a Chinese single institution (CMU-SO). Patient survival was analyzed using Kaplan-Meier survival curves, and comparisons were performed using the log-rank test. MUC occurred more frequently in patients who exhibited higher pT category, higher pN category, higher TNM stage, right-sided colon cancer, and higher histological grade. Based on SEER dataset, MUC was an independent negative survival indicator in rectal cancer (HR 1.125, 1.056-1.199; P<0.001). However, there was no significant difference in left-sided colon cancer (P>1.000). In particular, MUC was an independent protective survival indicator in right-sided colon cancer (HR 0.925, 0.888-0.962; P<0.001).
Conclusions
MUC was independently associated with poorer outcome for rectal cancer and was an independent protective survival indicator in right-sided colon cancer. MUC exhibited a different outcome relative to tumor position for patients with colorectal cancer.
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To analyze a series of cases of osteosarcoma of the jaw.
Methods and Results
The study comprised 74 cases of osteosarcoma of the jaw. Their clinical, radiographic and histopathological features were analyzed and their frequency with respect to aggressive and malignant pathologies of the jaw was determined. Survival was assessed in 17 cases with available follow-up. Osteosarcoma of the jaw accounted for 10% of primary malignant and aggressive tumors of the jaws, and for 8% of all malignant lesions of the jaw, including metastatic and lymphoproliferative tumors. Mean age was 43±18 years. Radiographic features varied greatly, were non-specific, with a predominance of mixed images. The dominant histological pattern was osteoblastic (48.5%), followed by chondroblastic pattern (37.1%). Survival at 5 years was 68%. Females and patients with a predominantly chondroblastic pattern had lower survival rates.
Conclusions
Osteosarcoma of the jaw was the most frequent primary malignant tumor of the jaw. Female gender and a predominantly chondroblastic pattern may be associated with a worse prognosis.
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Mammary analogue secretory carcinoma (MASC) of salivary glands shows morphological similarities with milk-secreting mammary epithelial cells. The aim of this study was to analyze immunohistochemical expression of adipophilin (a component of milk lipid globule membranes) and of proteins related to secretory mechanisms (STAT5a and mammaglobin) in MASC and other salivary tumors.
Methods and Results
Ten cases of MASC (all with ETV6 translocation) and 83 other salivary carcinomas were studied. In all cases of MASC, adipophilin stained numerous large lipid droplets. These droplets were minute in other salivary carcinomas, except for sebaceous carcinoma. Overexpression of STAT5a was detected in all cases of MASC but only occasionally in other carcinomas. Mammaglobin expression occurred mainly in MASC (70% of cases), whereas in other carcinomas it was uncommon and limited. Only MASC showed cytoplasmic reactivity for p63, particularly in papillary-cystic areas. Positivity for S100, vimentin and high-molecular-weight keratin was observed in 100% of MASC cases.
Conclusions
MASC is a lipid-rich tumor containing large lipid droplets covered by adipophilin. This finding can be included among its immunohistochemical defining features and possibly represents a lactational-like secretory differentiation. Strong expression of STAT5a and cytoplasmic p63 in MASC reinforces this hypothesis.
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Background:
Operation of the immune system is multivariate. Reduction of the dimensionality is essential to facilitate understanding of this complex biological system. One multi-dimensional facet of the immune system is the binding of epitopes to the MHC-I and MHC-II molecules by diverse populations of individuals. Prediction of such epitope binding is critical and several immunoinformatic strategies utilizing amino acid substitution matrices have been designed to develop predictive algorithms. Contemporaneously, computational and statistical tools have evolved to handle multivariate and megavariate analysis, but these have not been systematically deployed in prediction of MHC binding. Partial least squares analysis, principal component analysis, and associated regression techniques have become the norm in handling complex datasets in many fields. Over two decades ago Wold and colleagues showed that principal components of amino acids could be used to predict peptide binding to cellular receptors. We have applied this observation to the analysis of MHC binding, and to derivation of predictive methods applicable on a whole proteome scale.
Results:
We show that amino acid principal components and partial least squares approaches can be utilized to visualize the underlying physicochemical properties of the MHC binding domain by using commercially available software. We further show the application of amino acid principal components to develop both linear partial least squares and non-linear neural network regression prediction algorithms for MHC-I and MHC-II molecules. Several visualization options for the output aid in understanding the underlying physicochemical properties, enable confirmation of earlier work on the relative importance of certain peptide residues to MHC binding, and also provide new insights into differences among MHC molecules. We compared both the linear and non-linear MHC binding prediction tools to several predictive tools currently available on the Internet.
Conclusions:
As opposed to the highly constrained user-interaction paradigms of web-server approaches, local computational approaches enable interactive analysis and visualization of complex multidimensional data using robust mathematical tools. Our work shows that prediction tools such as these can be constructed on the widely available JMP® platform, can operate in a spreadsheet environment on a desktop computer, and are capable of handling proteome-scale analysis with high throughput.
Background:
One of the major challenges in the field of vaccine design is to predict conformational B-cell epitopes in an antigen. In the past, several methods have been developed for predicting conformational B-cell epitopes in an antigen from its tertiary structure. This is the first attempt in this area to predict conformational B-cell epitope in an antigen from its amino acid sequence.
Results:
All Support vector machine (SVM) models were trained and tested on 187 non-redundant protein chains consisting of 2261 antibody interacting residues of B-cell epitopes. Models have been developed using binary profile of pattern (BPP) and physiochemical profile of patterns (PPP) and achieved a maximum MCC of 0.22 and 0.17 respectively. In this study, for the first time SVM model has been developed using composition profile of patterns (CPP) and achieved a maximum MCC of 0.73 with accuracy 86.59%. We compare our CPP based model with existing structure based methods and observed that our sequence based model is as good as structure based methods.
Conclusion:
This study demonstrates that prediction of conformational B-cell epitope in an antigen is possible from is primary sequence. This study will be very useful in predicting conformational B-cell epitopes in antigens whose tertiary structures are not available. A web server CBTOPE has been developed for predicting B-cell epitope http://www.imtech.res.in/raghava/cbtope/.
Background:
Innate immunity is the first line of defence offered by host cells to infections. Macrophage cells involved in innate immunity are stimulated by lipopolysaccharide (LPS), found on bacterial cell surface, to express a complex array of gene products. Persistent LPS stimulation makes a macrophage tolerant to LPS with down regulation of inflammatory genes ("pro-inflammatory") while continually expressing genes to fight the bacterial infection ("antibacterial"). Interactions of transcription factors (TF) at their cognate TF binding sites (TFBS) on the expressed genes are important in transcriptional regulatory networks that control these pro-inflammatory and antibacterial expression paradigms involved in LPS stimulation.
Results:
We used differential expression patterns in a public domain microarray data set from LPS-stimulated macrophages to identify 228 pro-inflammatory and 18 antibacterial genes. Employing three different motif search tools, we predicted respectively four and one statistically significant TF-TFBS interactions from the pro-inflammatory and antibacterial gene sets. The biological literature was utilized to identify target genes for the four pro-inflammatory profile TFs predicted from the three tools, and 18 of these target genes were observed to follow the pro-inflammatory expression pattern in the original microarray data.
Conclusions:
Our analysis distinguished pro-inflammatory vs. antibacterial transcriptomic signatures that classified their respective gene expression patterns and the corresponding TF-TFBS interactions in LPS-stimulated macrophages. By doing so, this study has attempted to characterize the temporal differences in gene expression associated with LPS tolerance, a major immune phenomenon implicated in various pathological disorders.
Background:
Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4+ T cells are needed for optimal CD8+ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4+ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4+ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy.
Results:
By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4+ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4+ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4+ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies.
Conclusions:
The identification of CD4+ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.
Background:
Improving our understanding of the immune response is fundamental to developing strategies to combat a wide range of diseases. We describe an integrated epitope analysis system which is based on principal component analysis of sequences of amino acids, using a multilayer perceptron neural net to conduct QSAR regression predictions for peptide binding affinities to 35 MHC-I and 14 MHC-II alleles.
Results:
The approach described allows rapid processing of single proteins, entire proteomes or subsets thereof, as well as multiple strains of the same organism. It enables consideration of the interface of diversity of both microorganisms and of host immunogenetics. Patterns of binding affinity are linked to topological features, such as extracellular or intramembrane location, and integrated into a graphical display which facilitates conceptual understanding of the interplay of B-cell and T-cell mediated immunity.Patterns which emerge from application of this approach include the correlations between peptides showing high affinity binding to MHC-I and to MHC-II, and also with predicted B-cell epitopes. These are characterized as coincident epitope groups (CEGs). Also evident are long range patterns across proteins which identify regions of high affinity binding for a permuted population of diverse and heterozygous HLA alleles, as well as subtle differences in reactions with MHCs of individual HLA alleles, which may be important in disease susceptibility, and in vaccine and clinical trial design. Comparisons are shown of predicted epitope mapping derived from application of the QSAR approach with experimentally derived epitope maps from a diverse multi-species dataset, from Staphylococcus aureus, and from vaccinia virus.
Conclusions:
A desktop application with interactive graphic capability is shown to be a useful platform for development of prediction and visualization tools for epitope mapping at scales ranging from individual proteins to proteomes from multiple strains of an organism. The possible functional implications of the patterns of peptide epitopes observed are discussed, including their implications for B-cell and T-cell cooperation and cross presentation.
A phylogeographic analysis of gene sequences important in determining body size in dogs, recently published in BMC Biology, traces the appearance of small body size to the Neolithic Middle East. This finding strengthens the association of this event with the development of sedentary societies, and perhaps even has implications for the inception of human social inequality.See research article http://www.biomedcentral.com/1741-7007/8/16/
Understanding the spatio-temporal subversion of host cell signaling by bacterial virulence factors is key to combating infectious diseases. Following a recent study by Buntru and co-workers published in BMC Biology, we review how fluorescence (Forster) resonance energy transfer (FRET) has been applied to studying host-pathogen interactions and consider the prospects for its future application.See research article http://www.biomedcentral.com/1741-7007/7/81.
A recent study in BMC Evolutionary Biology has shown that genetically similar individual ring-tailed lemurs are also more similar in their scent composition, suggesting a possible mechanism of kin recognition. Theoretical and experimental studies reveal challenges ahead in achieving a true systems-level understanding of this process and its outcomes.See research article http://www.biomedcentral.com/1471-2148/9/281.
Acoel and platyhelminth worms are particularly attractive invertebrate models for stem-cell research because their bodies are continually renewed from large pools of somatic stem cells. Several recent studies, including one in BMC Developmental Biology, are beginning to reveal the cellular dynamics and molecular basis of stem-cell function in these animals.See research article http://www.biomedcentral.com/1471-213X/9/69.
Two recent studies in BMC Biology and Evolution raise important questions about a textbook case of frequency-dependent selection in scale-eating cichlid fishes. They also suggest a fascinating new line of research testing the effects of handed behavior on morphological asymmetry.See research article http://www.biomedcentral.com/1741-7007/8/8.
Background:
Most biomedical corpora have not been used outside of the lab that created them, despite the fact that the availability of the gold-standard evaluation data that they provide is one of the rate-limiting factors for the progress of biomedical text mining. Data suggest that one major factor affecting the use of a corpus outside of its home laboratory is the format in which it is distributed. This paper tests the hypothesis that corpus refactoring – changing the format of a corpus without altering its semantics – is a feasible goal, namely that it can be accomplished with a semi-automatable process and in a time-effcient way. We used simple text processing methods and limited human validation to convert the Protein Design Group corpus into two new formats: WordFreak and embedded XML. We tracked the total time expended and the success rates of the automated steps.
Results:
The refactored corpus is available for download at the BioNLP SourceForge website http://bionlp.sourceforge.net. The total time expended was just over three person-weeks, consisting of about 102 hours of programming time (much of which is one-time development cost) and 20 hours of manual validation of automatic outputs. Additionally, the steps required to refactor any corpus are presented.
Conclusion:
We conclude that refactoring of publicly available corpora is a technically and economically feasible method for increasing the usage of data already available for evaluating biomedical language processing systems.
Nanotechnology research has lately been of intense interest because of its perceived potential for many diverse fields of science. Nanotechnology's tools have found application in diverse fields, from biology to device physics. By the 1990s, there was a concerted effort in the United States to develop a national initiative to promote such research. The success of this effort led to a significant influx of resources and interest in nanotechnology and nanobiotechnology and to the establishment of centralized research programs and facilities. Further government initiatives (at federal, state, and local levels) have firmly cemented these disciplines as 'big science,' with efforts increasingly concentrated at select laboratories and centers. In many respects, these trends mirror certain changes in academic science over the past twenty years, with a greater emphasis on applied science and research that can be more directly utilized for commercial applications.We also compare the National Nanotechnology Initiative and its successors to the Human Genome Project, another large-scale, government funded initiative. These precedents made acceptance of shifts in nanotechnology easier for researchers to accept, as they followed trends already established within most fields of science. Finally, these trends are examined in the design of technologies for detection and treatment of cancer, through the Alliance for Nanotechnology in Cancer initiative of the National Cancer Institute. Federal funding of these nanotechnology initiatives has allowed for expansion into diverse fields and the impetus for expanding the scope of research of several fields, especially biomedicine, though the ultimate utility and impact of all these efforts remains to be seen.
Background:
Collaborative efforts of physicians and basic scientists are often necessary in the investigation of complex disorders. Difficulties can arise, however, when large amounts of information need to reviewed. Advanced information retrieval can be beneficial in combining and reviewing data obtained from the various scientific fields. In this paper, a team of investigators with varying backgrounds has applied advanced information retrieval methods, in the form of text mining and entity relationship tools, to review the current literature, with the intention to generate new insights into the molecular mechanisms underlying a complex disorder. As an example of such a disorder the Complex Regional Pain Syndrome (CRPS) was chosen. CRPS is a painful and debilitating syndrome with a complex etiology that is still unraveled for a considerable part, resulting in suboptimal diagnosis and treatment.
Results:
A text mining based approach combined with a simple network analysis identified Nuclear Factor kappa B (NFκB) as a possible central mediator in both the initiation and progression of CRPS.
Conclusion:
The result shows the added value of a multidisciplinary approach combined with information retrieval in hypothesis discovery in biomedical research. The new hypothesis, which was derived in silico, provides a framework for further mechanistic studies into the underlying molecular mechanisms of CRPS and requires evaluation in clinical and epidemiological studies.
Data management and integration are complicated and ongoing problems that will require commitment of resources and expertise from the various biological science communities. Primary components of successful cross-scale integration are smooth information management and migration from one context to another. We call for a broadening of the definition of bioinformatics and bioinformatics training to span biological disciplines and biological scales. Training programs are needed that educate a new kind of informatics professional, Biological Information Specialists, to work in collaboration with various discipline-specific research personnel. Biological Information Specialists are an extension of the informationist movement that began within library and information science (LIS) over 30 years ago as a professional position to fill a gap in clinical medicine. These professionals will help advance science by improving access to scientific information and by freeing scientists who are not interested in data management to concentrate on their science.
Background:
Biological organisms and their components are better conceived within categories based on similarity rather than on identity. Biologists routinely operate with similarity-based concepts such as "model organism" and "motif." There has been little exploration of the characteristics of the similarity-based categories that exist in biology. This study uses the case of the discovery and classification of zinc finger proteins to explore how biological categories based in similarity are represented.
Results:
The existence of a category of "zinc finger proteins" was based in 1) a lumpy gradient of similarity, 2) a link between function and structure, 3) establishment of a range of appearance across systems and organisms, and 4) an evolutionary locus as a historically based common-ground.
Conclusion:
More systematic application of the idea of similarity-based categorization might eliminate the assumption that biological characteristics can only contribute to narrow categorization of humans. It also raises possibilities for refining data-driven exploration efforts.
Blood chimaera is a rare but important issue for immunohaematology laboratories. Several molecular approaches, such as ABO genotyping, human leucocyte antigen (HLA) typing and DNA short tandem repeat (STR) analysis, have been used to identify chimaerism. Unfortunately, the minor allele population can be overlooked by PCR-based methods, which preferentially amplify the major allele population. A case with AweakB (AwB), demonstrating a mixed-field pattern, was sent to our laboratory for further evaluation. Direct sequencing of ABO exons 6 and 7 revealed a B101/O02 genotype. Analysis of the 12 STR loci and HLA typing did not provide any evidence of chimaerism. However, amplification refractory mutation system (ARMS)-PCR identified the minor A102 allele in addition to B101/O02. Three alleles of the chimaera were confirmed by cloning and sequencing. Thus, ARMS-PCR is essential, especially in the case of a chimaera with a minor allele population.
BRAF V600R-M-D are uncommon mutations, not included in the experimental protocols of BRAF selective inhibitors. We report the evaluation of correlations among different types of BRAF somatic mutations in melanoma and their management with BRAF inhibitors. 21 patients with BRAF mutated metastatic melanoma were enrolled in the protocol with BRAF inhibitors for compassionate use at the University of Modena. Hot spot V600E mutations were found in 19 patients. V600R mutation and double (V600E -V600M) mutation were identified in two melanomas. In one case, V600K mutation was found. Two screening failures were noted. Mean progression free survival at follow-up of to 8 weeks, was 7.6 months. Five patients had a very short follow-up and the experimental protocol is still ongoing, so we cannot provide complete follow-up data. However, all of them are still under treatment and disease progression free. An objective response with few side effects was observed in all patients. in vitro studies with the aim of testing drug sensitivity.
There are many causes of raised serum ferritin concentrations including iron overload, inflammation and liver disease to name but a few examples. Cases of extreme hyperferritinaemia (serum ferritin concentration equal to or greater than 10 000 ug/l) are being reported in laboratories but the causes of this are unclear. We conducted an audit study to explore this further. Extreme hyperferritinaemia was rare with only 0.08% of ferritin requests displaying this. The main causes of extreme hyperferritinaemia included multiple blood transfusions, malignant disease, hepatic disease and suspected Still's disease.
Advances in medical laboratory technology have driven major changes in the practice of laboratory medicine over the past two decades by the development of automated, cross-disciplinary single platform analysers. This has led to the blurring of boundaries between traditional disciplines and the emergence of core automated or blood science laboratories. This paper was commissioned by the Union of European Medical Specialists to examine the changing role of laboratory-based physicians in the light of these advances by focusing on the added value of expert interpretation of test results and resultant improvements in clinical outcomes. The paper also considers the broad range of responsibilities of laboratory-based physicians and the difficulties in precisely measuring how this translates into improved clinical outcomes. Given its provenance, the paper concentrates predominantly on the role of laboratory-based physicians while acknowledging the essential and vital role of scientists in running diagnostic laboratory services.
We report a case with a history of occupational asbestos exposure in which malignant pleural mesothelioma (MPM) was suspected clinically and diagnosed postmortem as pleural involvement of extrapulmonary small cell carcinoma (SCC). An 85-year-old man with a 65 pack-year history of smoking was referred to our hospital in June 2011. The patient had been exposed to asbestos in the iron production industry over the course of 30 years, and an irregular thickening of the right pleura was observed on chest CT at a medical check-up. The patient had a history of chronic hepatitis C and had been undergone transurethral resection for urothelial bladder cancer five times since 2006. Chest CT revealed neoplastic thickening of the right pleura, which had grown over 6 months (figure 1). The CT scan demonstrated bilateral pleural plaques, but no mass-like lesion in other organs, including the lungs, or mediastinal lymphadenopathy. The patient was...
Stage-specific expression of dynein light chain-1 and its interacting kinase, p21-activated kinase-1, in rodent testes: implications in spermiogenesis. Rui-An Wang, Ming Zhao, Marvin L. Meistrich, and Rakesh Kumar. J Histochem Cytochem. 53(10):1235–1243. DOI:10.1369/jhc.5A6688.2005.
BUBR1 (budding uninhibited by benzimidazole-related 1) represents the component of a controlling complex in mitosis. Defects in mitotic control complex result in chromosomal instability and, as a result, disturb the mitotic process. This study was aimed at examining the prognostic value linked to the expression of BUBR1 in a group of patients with breast cancer. We analyzed the expression of BUBR1 in 98 stage II breast cancer patients with a median follow-up of 15 years. Immunohistochemical reactions were performed using monoclonal antibodies against BUBR1. We also studied the prognostic value of BUBR1 mRNA expression using the Kaplan-Meier (KM) plotter, which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. A background database was established using gene expression data and survival information on 2422 patients downloaded from the Gene Expression Omnibus (GEO; Affymetrix HGU133A and HGU133+2 microarrays). The median relapse-free survival was 6.43 years. Univariate and multivariate analyses showed that higher expression of BUBR1 was typical for cases of shorter overall survival, disease-free time, and disease-specific survival. KM plotter analysis showed that elevated BUBR1 mRNA expression had a negative impact on patients’ relapse-free, distant metastases–free, and overall survival. Elevated BUBR1 expression was associated with poor survival in early stage breast cancer patients.
We made a qualitative and quantitative comparison between a state-of-the-art implementation of micro-Computed Tomography (microCT) and the scanning Thin-Sheet Laser Imaging Microscopy (sTSLIM) method, applied to mouse cochleae. Both imaging methods are non-destructive and perform optical sectioning, respectively, with X-rays and laser light. MicroCT can be used on fresh or fixed tissue samples and is primarily designed to image bone rather than soft tissues. It requires complex back-projection algorithms to produce a two-dimensional image, and it is an expensive instrument. sTSLIM requires that a specimen be chemically fixed, decalcified, and cleared; but it produces high-resolution images of soft and bony tissues with minimum image postprocessing and is less expensive than microCT. In this article, we discuss the merits and disadvantages of each method individually and when combined.
We analyze the effect of chronic undernourishment on extensor digitorum longus (EDL) muscle maturation in the rat. Cytochrome c oxidase (COX) and alkaline ATPase histoenzymatic techniques were used to determine the relative proportion of different fiber types (oxidative/glycolytic and type I, IIa/IId, or IIb, respectively) and their cross-sectional area in control and undernourished EDL muscles at several postnatal (PN) ages. From PN days 15 to 45, undernourished EDL muscles showed predominance of oxidative and type IIa/IId fibers, but from PN days 60 to 90, there were a larger proportion of oxidative fibers and an equal proportion of type IIa/IId and IIb fibers. Meanwhile, in adult stages (from PN days 130–365), the relative proportion of fiber types in control and undernourished EDL muscles showed no significant differences. In addition, from PN days 15 to 90, there was a significant reduction in the cross-sectional area of all fibers (slow: 13–53%; intermediate: 24–74%; fast: 9–80%) but no differences from PN days 130 to 365. It is suggested that chronic undernourishment affects the maturation of fast-type muscle fibers only at juvenile stages (from PN days 15–45) and the probable occurrence of adaptive mechanisms in muscle fibers, allowing adult rats to counterbalance the alterations provoked by chronic food deprivation.
Endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2), a new splicing variant of VEGFR-2, was shown to be the first endogenous specific inhibitor of lymphatic vessel growth. The expression of esVEGFR-2 and its clinicopathological roles in esophageal squamous cell carcinoma (ESCC) are unclear. In this article, quantitative RT-PCR was employed to detect the mRNA levels of esVEGFR-2 and VEGF-C in 90 paired primary ESCC tissues, along with immunohistochemical staining to measure esVEGFR-2 protein in 182 ESCC primary tissues. Correlations between esVEGFR-2 expression and clinicopathological features were also analyzed. Compared with the corresponding non-neoplastic esophageal mucosa tissues, the mRNA level of esVEGFR-2 was decreased, whereas the mRNA level of VEGF-C was increased in ESCCs. Downregulation of esVEGFR-2 mRNA level was significantly correlated with pTNM stages (2 = 7.790, p=0.02). Immunohistochemical staining of esVEGFR-2 was inclined to be reduced in ESCC tissues; lower esVEGFR-2 protein expression was related to better prognosis (2 = 6.366, p=0.012), whereas higher esVEGFR-2 protein accumulation in ESCC tissues was an independent prognostic factor for poor survival of patients (hazard ratio, 1.606; 95% confidence interval, 1.042–2.476; p=0.032). Taken together, altered expression of esVEGFR-2 is correlated with progression of ESCC. esVEGFR-2 might serve as a new independent prognostic marker for ESCC patients.
Stage-specific expression of dynein light chain-1 and its interacting kinase, p21-activated kinase-1, in rodent testes: implications in spermiogenesis. Rui-An Wang, Ming Zhao, Marvin L. Meistrich, and Rakesh Kumar. J Histochem Cytochem. 53(10):1235–1243. DOI:10.1369/jhc.5A6688.2005.
BUBR1 (budding uninhibited by benzimidazole-related 1) represents the component of a controlling complex in mitosis. Defects in mitotic control complex result in chromosomal instability and, as a result, disturb the mitotic process. This study was aimed at examining the prognostic value linked to the expression of BUBR1 in a group of patients with breast cancer. We analyzed the expression of BUBR1 in 98 stage II breast cancer patients with a median follow-up of 15 years. Immunohistochemical reactions were performed using monoclonal antibodies against BUBR1. We also studied the prognostic value of BUBR1 mRNA expression using the Kaplan-Meier (KM) plotter, which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. A background database was established using gene expression data and survival information on 2422 patients downloaded from the Gene Expression Omnibus (GEO; Affymetrix HGU133A and HGU133+2 microarrays). The median relapse-free survival was 6.43 years. Univariate and multivariate analyses showed that higher expression of BUBR1 was typical for cases of shorter overall survival, disease-free time, and disease-specific survival. KM plotter analysis showed that elevated BUBR1 mRNA expression had a negative impact on patients’ relapse-free, distant metastases–free, and overall survival. Elevated BUBR1 expression was associated with poor survival in early stage breast cancer patients.
We made a qualitative and quantitative comparison between a state-of-the-art implementation of micro-Computed Tomography (microCT) and the scanning Thin-Sheet Laser Imaging Microscopy (sTSLIM) method, applied to mouse cochleae. Both imaging methods are non-destructive and perform optical sectioning, respectively, with X-rays and laser light. MicroCT can be used on fresh or fixed tissue samples and is primarily designed to image bone rather than soft tissues. It requires complex back-projection algorithms to produce a two-dimensional image, and it is an expensive instrument. sTSLIM requires that a specimen be chemically fixed, decalcified, and cleared; but it produces high-resolution images of soft and bony tissues with minimum image postprocessing and is less expensive than microCT. In this article, we discuss the merits and disadvantages of each method individually and when combined.
We analyze the effect of chronic undernourishment on extensor digitorum longus (EDL) muscle maturation in the rat. Cytochrome c oxidase (COX) and alkaline ATPase histoenzymatic techniques were used to determine the relative proportion of different fiber types (oxidative/glycolytic and type I, IIa/IId, or IIb, respectively) and their cross-sectional area in control and undernourished EDL muscles at several postnatal (PN) ages. From PN days 15 to 45, undernourished EDL muscles showed predominance of oxidative and type IIa/IId fibers, but from PN days 60 to 90, there were a larger proportion of oxidative fibers and an equal proportion of type IIa/IId and IIb fibers. Meanwhile, in adult stages (from PN days 130–365), the relative proportion of fiber types in control and undernourished EDL muscles showed no significant differences. In addition, from PN days 15 to 90, there was a significant reduction in the cross-sectional area of all fibers (slow: 13–53%; intermediate: 24–74%; fast: 9–80%) but no differences from PN days 130 to 365. It is suggested that chronic undernourishment affects the maturation of fast-type muscle fibers only at juvenile stages (from PN days 15–45) and the probable occurrence of adaptive mechanisms in muscle fibers, allowing adult rats to counterbalance the alterations provoked by chronic food deprivation.
Endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2), a new splicing variant of VEGFR-2, was shown to be the first endogenous specific inhibitor of lymphatic vessel growth. The expression of esVEGFR-2 and its clinicopathological roles in esophageal squamous cell carcinoma (ESCC) are unclear. In this article, quantitative RT-PCR was employed to detect the mRNA levels of esVEGFR-2 and VEGF-C in 90 paired primary ESCC tissues, along with immunohistochemical staining to measure esVEGFR-2 protein in 182 ESCC primary tissues. Correlations between esVEGFR-2 expression and clinicopathological features were also analyzed. Compared with the corresponding non-neoplastic esophageal mucosa tissues, the mRNA level of esVEGFR-2 was decreased, whereas the mRNA level of VEGF-C was increased in ESCCs. Downregulation of esVEGFR-2 mRNA level was significantly correlated with pTNM stages (2 = 7.790, p=0.02). Immunohistochemical staining of esVEGFR-2 was inclined to be reduced in ESCC tissues; lower esVEGFR-2 protein expression was related to better prognosis (2 = 6.366, p=0.012), whereas higher esVEGFR-2 protein accumulation in ESCC tissues was an independent prognostic factor for poor survival of patients (hazard ratio, 1.606; 95% confidence interval, 1.042–2.476; p=0.032). Taken together, altered expression of esVEGFR-2 is correlated with progression of ESCC. esVEGFR-2 might serve as a new independent prognostic marker for ESCC patients.
Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.
We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.
We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.
A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.
A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.
The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.
Medical Immunology will be publishing invited Reviews and Commentaries from investigators who are at the forefront of their fields, to up-date our readers as to the current state of their art. These Reviews and Commentaries will be accompanied by Editorials that place the current work into the perspective of the first contribution in an area, which resulted in a "Classic" paper. Where possible, links will be provided to the original publication, so that the modern student of immunology can read the original and draw their own conclusions as to the value of the "Classic" contribution, and its relationship to our contemporary views as to how the immune system functions. To begin this process at the very dawn of immunology, we highlight Sir Edward Jenner's first descriptions of the use of cowpox to immunize individuals against the dread disease smallpox.
Eradication of the smallpox virus through extensive global vaccination efforts has resulted in one of the most important breakthroughs in medical history, saving countless lives from the severe morbidity and mortality that is associated with this disease. Although smallpox is now extinct in nature, laboratory stocks of this virus still remain and the subject of smallpox vaccination has gained renewed attention due to the potential risk that smallpox may be used as a biological weapon by terrorists or rogue states. Despite having the longest history of any modern vaccine, there is still much to be learned about smallpox vaccination and the correlates of protection remain to be formally defined. This Commentary will discuss the strengths and weaknesses of traditional smallpox vaccination in comparison with immunization using modified vaccinia virus Ankura (MVA), a non-replicating virus with a strong safety record but weakened immunogenicity.
FADD (Fas Associated protein with Death Domain) is a key adaptor molecule transmitting the death signal mediated by death receptors. In addition, this multiple functional protein is implicated in survival/proliferation and cell cycle progression. FADD functions are regulated via cellular sublocalization, protein phosphorylation, and inhibitory molecules. In the present review, we focus on the role of the FADD adaptor in cancer. Increasing evidence shows that defects in FADD protein expression are associated with tumor progression both in mice and humans. Better knowledge of the mechanisms leading to regulation of FADD functions will improve understanding of tumor growth and the immune escape mechanisms, and could open a new field for therapeutic interventions.
It has been more than 100 years since the realization that microbes are capable of causing disease. In that time, we have learned a great deal as to how each organism has adapted to the immune system so as to avoid elimination. As well, we have also learned an immense amount since Louis Pasteur first proposed that the solution to infectious diseases was to culture the microbes and attenuate their virulence, so as to use them as vaccines. From the optimism and promise of the 19th century and immunization as the ultimate answer to the invasion by the microbial world, to the scientific realities of the 21st century, it is of interest to retrace the steps of the earliest microbiologists cum immunologists, to realize how far we've come, as well as how far we yet have to go. This editorial focuses on the history of anthrax as a microbial disease, and the earliest efforts at producing a vaccine for its prevention.
The research results presented in this paper are part of a larger study on the materials and techniques used in polychrome altarpieces of gilded woodcarving decoration (“talha douradaâ€) in Portugal. The paper focuses on a narrative Portuguese Altarpiece from Miranda do Douro, considered one of the masterpieces of “talha dourada†among all the retables of the Iberian Peninsula in XVIIth and XVIIIth centuries. Although on the Portuguese territory, the altarpiece was made by artists from the Royal Spanish school of Valladolid, under a mannerist style. Thus the study opens a window on the artists' circulation between Spain and Portugal and influences of the Spanish schools in Baroque epoch on the Portuguese “talhaâ€. During its history this altarpiece underwent several transformations and extensive conservation treatments in 1989. On this occasion more than 50 samples were collected and analyzed using an interdisciplinary multi-technique methodology. 27 of these samples are chosen for this study in order to investigate the chromatic palette, the materials and techniques used in the polychromy of the retable. A novel protocol of investigation using different conventional and unconventional analytical techniques (OM + fluorescent staining tests on cross-sections, Raman microscopy, XRD, XRF, X-ray micro-CT, SEM-EDX, MALDI-TOF-MS and LC-MS/MS) was established within an innovative research project (http://sites.fct.unl.pt/gilt-teller/) and applied on these samples. This protocol is necessary to confirm the results obtained in the 1989 campaign and to have further insight into the gilding and polychrome decoration materials and techniques and the additional information reported in the historical documents. The material and technical history of this important altarpiece will be thus re-documented from a scientific perspective, meant to confirm and bring new information on the decorative technique used in the creation of this complex Portuguese monument.
Chromophobe hepatocellular carcinoma with abrupt anaplasia: a proposal for a new subtype of hepatocellular carcinoma with unique morphological and molecular features
Authors: Laura D Wood, Christopher M Heaphy, Hubert Darius-J Daniel, Bita V Naini, Charles R Lassman, May R Arroyo, Ihab R Kamel, David P Cosgrove, John K Boitnott, Alan K Meeker
& Michael S Torbenson
Authors: Wenlei He, John C Cheville, Peter M Sadow, Anuradha Gopalan, Samson W Fine, Hikmat A Al-Ahmadie, Ying-Bei Chen, Esther Oliva, Paul Russo, Victor E Reuter
& Satish K Tickoo
KRAS mutant allele-specific imbalance is associated with worse prognosis in pancreatic cancer and progression to undifferentiated carcinoma of the pancreas
MicroRNAs(miRNA) are noncoding RNAs of about 19--23 nucleotides that are crucial for many biological processes. Members of the microRNA-148/152(miR-148/152) family, which include microRNA-148a(miR-148a), microRNA-148b(miR-148b), and microRNA-152(miR-152), are expressed differently in tumor and nontumor tissues and are involved in the genesis and development of disease. Furthermore, members of the miR-148/152 family are important in the growth and development of normal tissues. Members of the miR-148/152 family regulate target genes and are regulated by methylation of CPG islands. In this review, we report recent studies on the expression of members of the miR-148/152 family, methylation of CPG islands, and their target genes in different diseases, as well as in normal tissues.
Background:
It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-2/Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. We designed the BH3 alpha-helix mimetic JY-1-106 to engage the hydrophobic BH3-binding grooves on the surfaces of both Bcl-xL and Mcl-1.
Methods:
JY-1-106--protein complexes were studied using molecular dynamics (MD) simulations and the SILCS methodology. We have evaluated the in vitro effects of JY-1-106 by using a fluorescence polarization (FP) assay, an XTT assay, apoptosis assays, and immunoprecipitation and western-blot assays. A preclinical human cancer xenograft model was used to test the efficacy of JY-1-106 in vivo.
Results:
MD and SILCS simulations of the JY-1-106--protein complexes indicated the importance of the aliphatic side chains of JY-1-106 to binding and successfully predicted the improved affinity of the ligand for Bcl-xL over Mcl-1. Ligand binding affinities were measured via an FP assay using a fluorescently labeled Bak-BH3 peptide in vitro. Apoptosis induction via JY-1-106 was evidenced by TUNEL assay and PARP cleavage as well as by Bax--Bax dimerization. Release of multi-domain Bak from its inhibitory binding to Bcl-2/Bcl-xL and Mcl-1 using JY-1-106 was detected via immunoprecipitation (IP) western blotting.At the cellular level, we compared the growth proliferation IC50s of JY-1-106 and ABT-737 in multiple cancer cell lines with various Bcl-xL and Mcl-1 expression levels. JY-1-106 effectively induced cell death regardless of the Mcl-1 expression level in ABT-737 resistant solid tumor cells, whilst toxicity toward normal human endothelial cells was limited. Furthermore, synergistic effects were observed in A549 cells using a combination of JY-1-106 and multiple chemotherapeutic agents. We also observed that JY-1-106 was a very effective agent in inducing apoptosis in metabolically stressed tumors. Finally, JY-1-106 was evaluated in a tumor-bearing nude mouse model, and was found to effectively repress tumor growth. Strong TUNEL signals in the tumor cells demonstrated the effectiveness of JY-1-106 in this animal model. No significant side effects were observed in mouse organs after multiple injections.
Conclusions:
Taken together, these observations demonstrate that JY-1-106 is an effective pan-Bcl-2 inhibitor with very promising clinical potential.
Background:
Patients with familial adenomatous polyposis (FAP) are at increased risk for the development of colorectal cancer. Surgery and chemoprevention are the most effective means to prevent cancer development. Thymoquinone (TQ) is considered the main compound of the volatile Nigella sativa seed oil and has been reported to possess anticarcinogenic properties. In this study we evaluated the chemopreventive properties of TQ in a mouse model of FAP.
Methods:
APCMin mice were fed with chow containing 37.5 mg/kg or 375 mg/kg TQ for 12 weeks. H&E stained intestine tissue sections were assessed for tumor number, localization, size, and grade. Immunohistochemistry for beta-catenin, c-myc, Ki-67 and TUNEL-staining was performed to investigate TQ's effect on major colorectal cancer pathways. TQ's impact on GSK-3beta and beta-catenin were studied in RKO cells.
Results:
375 mg/kg but not 37.5 mg/kg TQ decreased the number of large polyps in the small intestine of APCMin mice. TQ induced apoptosis in the neoplastic tissue but not in the normal mucosa. Furthermore, upon TQ treatment, beta-catenin was retained at the membrane and c-myc decreased in the nucleus, which was associated with a reduced cell proliferation in the villi. In vitro, TQ activated GSK-3beta, which induced membranous localization of beta-catenin and reduced nuclear c-myc expression.
Conclusions:
In summary, TQ interferes with polyp progression in ApcMin mice through induction of tumor-cell specific apoptosis and by modulating Wnt signaling through activation of GSK-3beta. Nigella sativa oil (or TQ) might be useful as nutritional supplement to complement surgery and chemoprevention in FAP.
Background:
Co-Activator Arginine Methyltransferase 1(CARM1) is an Estrogen Receptor (ER) cofactor that remodels chromatin for gene regulation via methylation of Histone3. We investigated CARM1 levels and localization across breast cancer tumors in a cohort of patients of either European or African ancestry.
Methods:
We analyzed CARM1 levels using tissue microarrays with over 800 histological samples from 549 female cancer patients from the US and Nigeria, Africa. We assessed associations between CARM1 expression localized to the nucleus and cytoplasm for 11 distinct variables, including; ER status, Progesterone Receptor status, molecular subtypes, ethnicity, HER2+ status, other clinical variables and survival.
Results:
We found that levels of cytoplasmic CARM1 are distinct among tumor sub-types and increased levels are associated with ER-negative (ER-) status. Higher nuclear CARM1 levels are associated with HER2 receptor status. EGFR expression also correlates with localization of CARM1 into the cytoplasm. This suggests there are distinct functions of CARM1 among molecular tumor types. Our data reveals a basal-like subtype association with CARM1, possibly due to expression of Epidermal Growth Factor Receptor (EGFR). Lastly, increased cytoplasmic CARM1, relative to nuclear levels, appear to be associated with self-identified African ethnicity and this result is being further investigated using quantified genetic ancestry measures.
Conclusions:
Although it is known to be an ER cofactor in breast cancer, CARM1 expression levels are independent of ER. CARM1 has distinct functions among molecular subtypes, as is indicative of its sub-cellular localization and it may function in subtype etiology. These sub-cellular localization patterns, indicate a novel role beyond its ER cofactor function in breast cancer. Differential localization among ethnic groups may be due to ancestry-specific polymorphisms which alter the gene product.
Background:
The aryl hydrocarbon receptor (AhR) ligand 6-Formylindolo(3,2-b)carbazole (FICZ) has received increasing attention since its identification as an endogenous AhR ligand and a photoproduct of tryptophan. FICZ and its metabolites have been detected in human fluids. We recently reported that AhR promotes retinoic acid (RA)-induced granulocytic differentiation of HL-60 myeloblastic leukemia cells by restricting the nuclear abundance of the stem cell associated transcription factor Oct4. The standard clinical management of acute promyelocytic leukemia (APL) is differentiation induction therapy using RA. But RA is not effective for other myeloid leukemias, making the mechanism of RA-induced differentiation observed in a non-APL myeloid leukemia of interest. To our knowledge, this is the first study regarding the influence of FICZ on RA-induced differentiation in any type of leukemic blasts.
Methods:
Using flow cytometry and Western blotting assays, we determined the effects of FICZ on RA-induced differentiation of HL-60 human leukemia cells. All experiments were performed in triplicate. The groups RA and FICZ + RA were compared using the Paired-Samples T-Test. Western blot figures present the typical blots.
Results:
We demonstrate that FICZ enhances RA-induced differentiation, assessed by the expression of the membrane differentiation marker CD11b; cell cycle arrest; and the functional differentiation marker, inducible-oxidative metabolism. FICZ causes changes in signaling events that are known to drive differentiation, and notably augments the RA-induced sustained activation of the RAF/MEK/ERK axis of the mitogen-activated protein kinase (MAPK) cascade. FICZ also augments expression of the known MAPK signaling regulatory molecules c-Cbl, VAV1, pY458 p85 PI3K, Src-family kinases (SFKs), and IRF-1, a transcription factor associated with this putative signalsome that promotes RA-induced differentiation. Moreover, FICZ in combination with RA also increases expression of AhR and even more so of both Cyp1A2 and p47phox, which are known to be transcriptionally regulated by AhR. pY1021 PDGFRbeta, a marker associated with retinoic acid syndrome was also increased.
Conclusions:
Our data suggest that FICZ modulates intracellular signaling pathways and enhances RA-induced differentiation.
Background:
The constitutive androstane receptor (CAR, NR1I3) plays a key role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes.
Results:
The expression of CAR mRNA throughout the circadian rhythm is reported for the first time in phase with the clock gene Bmal1 and in antiphase with the clock-controlled gene Rev-erbα mRNAs, with a peak at Zeitgeber time (ZT) 20 and a trough at ZT8, and a peak/trough ratio of 2.0. The diurnal difference in CAR mRNA expression might underlie the 1.7-fold difference in the magnitude of the PB-dependent induction of CYP2B1/2 mRNA.
Conclusion:
The circadian oscillation of xenosensor gene CAR mRNA expression is partially responsible for chronopharmacokinetics and chronopharmacology in disease.
Background:
The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3–5%), the latter was carefully removed by ultrafiltration.
Results:
As previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780.
Conclusions:
These results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in intact cells that express ER, E2-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.
Background:
Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor α (RXRα), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRα are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRα was a common mechanism underlying the negative hepatic APR.
Results:
Nuclear RXRα protein levels were significantly reduced (~50%) within 1–2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRα was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRα partner, the retinoic acid receptor (RARα), was unaffected by LPS. A potential cell-signaling modulator of RXRα activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1–2 hours, coincident with maximal levels of cytoplasmic RXRα. RNA levels of RXRα were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRα were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRα-containing heterodimer pairs.
Conclusion:
The subcellular localization of native RXRα rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRα-regulated genes in inflammation.
Background:
Ligand-bound estrogen receptor α (ERα) and estrogen receptor β (ERβ) modulate AP-1-dependent transcription via protein-protein interactions on DNA, in a manner that depends on the type of cells and the subtype of ER. We present here evidence for an additional mechanism by which ERs modulate the transcriptional activity of AP-1.
Results:
We show that ERs located in the cytoplasm efficiently activate transcription at AP-1 sites in response to 17β-estradiol, while ERs present in the nucleus repress transcription under the same conditions. 17β-estradiol-induced activation of the coll-73-luc reporter correlated with cytoplasmic localization of various ERα and ERβ mutant receptors, and was inhibited in the presence of the full estrogen antagonist ICI 182,780 and the MAP-kinase inhibitor UO126. We also show that the selective estrogen receptor modulator (SERM) tamoxifen is as potent as 17β-estradiol in inducing activation of AP-1 when ERα is present in the cytoplasm.
Conclusions:
These results suggest that non-genomic signalling is involved in the mechanism by which ERα and ERβ influence AP-1-dependent transcription. We have previously shown that Stat3 and Stat5 are targeted by non-genomic actions of ERs, and the results presented here allow us to conclude that ERs bound to 17β-estradiol mediate the transcriptional activation of promoters regulated by AP-1 and by Stat proteins via different combinations of signal transduction pathways. Our observations thereby provide new insights into the mechanisms by which ERs act at alternate response elements, and suggest a mechanism by which tamoxifen exerts its action as a tissue-selective agonist.
Background:
Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals.
Results:
To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR.
Conclusion:
Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species.
The accuracy of pairing of the anticodon of the initiator tRNA (tRNAfMet) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m2G966 (methylated by RsmD), m5C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNAfMet. We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA (tRNAfMet with CAU anticodon); CAC and CAU with tRNA; UAG with tRNA; UAC with tRNA; and AUC with tRNA using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.
Protein-coding genes account for only a small part of the human genome, whereas the vast majority of transcripts make up the non-coding RNAs including long non-coding RNAs (lncRNAs). Accumulating evidence indicates that lncRNAs could play a critical role in regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. LncRNA loc285194 was previously shown to be within a tumor suppressor unit in osteosarcoma and to suppress tumor cell growth. However, it is unknown regarding the regulation of loc285194. Moreover, the underlying mechanism by which loc285194 functions as a potential tumor suppressor is elusive. In this study, we show that loc285194 is a p53 transcription target; ectopic expression of loc285194 inhibits tumor cell growth both in vitro and in vivo. Through deletion analysis, we identify an active region responsible for tumor cell growth inhibition within exon 4, which harbors two miR-211 binding sites. Importantly, this loc285194-mediated growth inhibition is in part due to specific suppression of miR-211. We further demonstrate a reciprocal repression between loc285194 and miR-211; in contrast to loc285194, miR-211 promotes cell growth. Finally, we detect downregulation of loc285194 in colon cancer specimens by quantitative PCR arrays and in situ hybridization of tissue microarrays. Together, these results suggest that loc285194 is a p53-regulated tumor suppressor, which acts in part through repression of miR-211.
The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNALeu recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.
The type II restriction endonuclease TseI recognizes the DNA target sequence 5'-G^CWGC-3' (where W = A or T) and cleaves after the first G to produce fragments with three-base 5'-overhangs. We have determined that it is a dimeric protein capable of cleaving not only its target sequence but also one containing A:A or T:T mismatches at the central base pair in the target sequence. The cleavage of targets containing these mismatches is as efficient as cleavage of the correct target sequence containing a central A:T base pair. The cleavage mechanism does not apparently use a base flipping mechanism as found for some other type II restriction endonuclease recognizing similarly degenerate target sequences. The ability of TseI to cleave targets with mismatches means that it can cleave the unusual DNA hairpin structures containing A:A or T:T mismatches formed by the repetitive DNA sequences associated with Huntington’s disease (CAG repeats) and myotonic dystrophy type 1 (CTG repeats).
Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.
Background:
Grafting procedures are an excellent tool to study long range signalling processes within a plant. In the last decade, suitable flat-surface grafting procedures for young Arabidopsis seedlings using a collar to support the graft have been developed, allowing the study of long-range signals from a molecular perspective.
Results:
In the modification presented here, scion and stock are put together on the medium without supporting elements, while cotyledons are removed from the scion, resulting in increased grafting success that can reach up to 100%. At the same time, the protocol enables to process as many as 36 seedlings per hour, which combined with the high success percentage represents increased efficiency per time unit.
Conclusions:
Growing cotyledons usually push the scion and the rootstock away in the absence of a supporting element. Removing them at the grafting step greatly improved success rate and reduced post-grafting manipulations.
Background:
Brassinosteriods (BRs), a group of important phytohormones, have various effects on plant growth and development. However, their physiological functions in plants have not been fully understood to date. Endogenous BRs in plant tissue are extremely low and the elucidation of BRs functions relies on sensitive detection method. Reported methods for the determination of BRs required large amount of plant tissue, tedious pretreatment process, and were lack of selectivity. Therefore, development of a simple and selective method for the sensitive quantification of BRs is highly needed.
Results:
We established a pretreatment method of BRs in plant tissues by employing double layered solid phase extraction (DL/SPE) combined with boronate affinity polymer monolith microextraction (BA/PMME). After the initial depigmentation with DL/SPE cartridge, boronate affinity polymer BA/PMME was employed to selectively extract BRs from sample matrix. Uniquely, most sample matrix was successfully removed by BA monolith purification. Using this method, BRs was determined by liquid chromatography-mass spectrometry (LC-MS). Endogenous active BRs could be detected in only 1 g fresh weigh (FW) leaves or 0.5 g FW flower tissues.
Conclusion:
A DL/SPE-BA/PMME pretreatment method for the determination of endogenous brassinosteroids in plant tissues was developed and validated. The proposed method was sensitive and selective. The proposed method may be further developed for the determination of other BRs including their precursors and conjugates.
Background From initial seed germination through reproduction, plants continuously reprogram their transcriptional repertoire to facilitate growth and development. This dynamic is mediated by a diverse but inextricably-linked catalog of regulatory proteins called transcription factors (TFs). Statistically quantifying TF binding site (TFBS) abundance in promoters of differentially expressed genes can be used to identify binding site patterns in promoters that are closely related to stress-response. Output from today's transcriptomic assays necessitates statistically-oriented software to handle large promoter-sequence sets in a computationally tractable fashion.Results We present Marina, an open-source software for identifying over-represented TFBSs from amongst large sets of promoter sequences, using an ensemble of 7 statistical metrics and binding-site profiles. Through software comparison, we show that Marina can identify considerably more over-represented plant TFBSs compared to a popular software alternative.Conclusions Marina was used to identify over-represented TFBSs in a two time-point RNA-Seq study exploring the transcriptomic interplay between soybean (Glycine max) and soybean rust (Phakopsora pachyrhizi). Marina identified numerous abundant TFBSs recognized by transcription factors that are associated with defense-response such as WRKY, HY5 and MYB2. Comparing results from Marina to that of a popular software alternative suggests that regardless of the number of promoter-sequences, Marina is able to identify significantly more over-represented TFBSs.
Background:
The hydraulic conductivity of the stem is a major factor limiting the capability of trees to transport water from the soil to transpiring leaves. During drought conditions, the conducting capacity of xylem can be reduced by some conduits being filled with gas, i.e. embolized. In order to understand the dynamics of embolism formation and repair, considerable attention has been given to developing reliable and accurate methods for quantifying the phenomenon. In the past decade, non-destructive imaging of embolism formation in living plants has become possible. Magnetic resonance imaging has been used to visualize the distribution of water within the stem, but in most cases it is not possible to resolve individual cells. Recently, high-resolution synchrotron x-ray microtomography has been introduced as a tool to visualize the water contents of individual cells in vivo, providing unprecedented insight into the dynamics of embolism repair. We have investigated the potential of an x-ray tube -based microtomography setup to visualize and quantify xylem embolism and embolism repair in water-stressed young saplings and shoot tips of Silver and Curly birch (Betula pendula and B. pendula var. carelica).
Results:
From the microtomography images, the water-filled versus gas-filled status of individual xylem conduits can be seen, and the proportion of stem cross-section that consists of embolized tissue can be calculated. Measuring the number of embolized vessels in the imaged area is a simple counting experiment. In the samples investigated, wood fibers were cavitated in a large proportion of the xylem cross-section shortly after watering of the plant was stopped, but the number of embolized vessels remained low several days into a drought period. Under conditions of low evaporative demand, also refilling of previously embolized conduits was observed.
Conclusions:
Desktop x-ray microtomography is shown to be an effective method for evaluating the water-filled versus embolized status of the stem xylem in a small living sapling. Due to its non-destructive nature, the risk of inducing embolisms during sampling is greatly reduced. Compared with synchrotron imaging beamlines, desktop microtomography offers easier accessibility, while maintaining sufficient resolution to visualize the water contents of individual cells.
Background:
Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems.
Results:
Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoViewTM FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells.
Conclusions:
Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists.
Spontaneous esophageal perforation into the pleural cavity (Boerhaave's syndrome) is a rare life-threatening condition, which requires early diagnosis and urgent management. The diagnosis of such critical condition in many cases is delayed because of atypical clinical presentation, resulting in increased morbidity and mortality. Cytological examination of pleural fluid can provide early, fast and accurate diagnosis of such critical condition and help in better and early management of this disease. We describe a case of an 81-year-old female with esophageal perforation who presented with a left sided pleural effusion. The correct diagnosis was established in this case by observing gastrointestinal-like fluid characteristics of the thoracic drainage upon cytological and chemical analyses and the rupture was confirmed by esophagography. The cytological examination of pleural fluid revealed benign reactive squamous cells, fungal organisms, bacterial colonies, and vegetable material consistent with a ruptured esophagus. Cytological examination of pleural fluid is a rapid and accurate technique that can help in establishing the diagnosis of this challenging entity and guide initiation proper management of this unusual entity.
Erika F Rodriguez, Sara E Monaco, Walid Khalbuss, R Marshall Austin, Liron Pantanowtiz
CytoJournal 2013 10(1):7-7
Intraperitoneal spread may occur with gynecological epithelial neoplasms, as well as with non-gynecological malignancies, which may result in serosal involvement with or without concomitant effusion. Therefore, washings in patients with abdominopelvic tumors represent important specimens for cytologic examination. They are primarily utilized for staging ovarian cancers, although their role has decreased in staging of endometrial and cervical carcinoma. Abdominopelvic washings can be positive in a variety of pathologic conditions, including benign conditions, borderline neoplastic tumors, locally invasive tumors, or distant metastases. In a subset of cases, washings can be diagnostically challenging due to the presence of co-existing benign cells (e.g., mesothelial hyperplasia, endosalpingiosis, or endometriosis), lesions in which there is only minimal atypia (e.g., serous borderline tumors) or scant atypical cells, and the rarity of specific tumor types (e.g., mesothelioma). Ancillary studies including immunocytochemistry and fluorescence in situ hybridization may be required in difficult cases to resolve the diagnosis. This article provides a comprehensive and contemporary review of abdominopelvic washings in the evaluation of gynecologic and non-gynecologic tumors, including primary peritoneal and mesothelial entities.
Brooke R Koltz, Donna K Russell, Naiji Lu, Thomas A Bonfiglio, Sharlin Varghese
CytoJournal 2013 10(1):6-6
Introduction: Automated screening of Thin Prep ® Papanicolaou Tests has become increasingly common in clinical practice. Increased productivity has initiated laboratory use of the Thin Prep ® Imaging System (TIS). Increased sensitivity is a potential additional benefit of TIS. Published studies have shown an increase in discovery of dysplastic cells. This study evaluates the effect of TIS on the incidence of atypical squamous cells high-grade squamous intraepithelial lesion not excluded (ASC-H) and high-grade squamous intraepithelial lesion (HGSIL) results on Thin Prep ® Pap Tests by comparing TIS-assisted and manual screening findings and the diagnoses on subsequent follow-up in a screening population over a 1-year time period. Materials and Methods: A compilation of all ASC-H and HGSIL cases was prepared by conducting a computerized search over a 1-year period (7/06-6/07). The accumulated cases include Thin Prep Pap tests that were both TIS and manually screened. Follow-up results of cytologic and histologic cervical specimens were obtained for a time period extending to 2010. Interpretation utilizing TIS was in place 10 months prior to the study's initiation. Results: During the study period 70,522 Pap tests were performed in our laboratory. One third (33%) of Pap tests were screened with assistance of TIS. Manual screening was performed on 47,380 Pap tests of which 153 (0.32%) were interpreted as ASC-H and 164 (0.35%) were interpreted as HGSIL. During the same time period automated screening (TIS) was performed on 23,111 Pap tests. Interpretation of 62 (0.27%) cases provided an ASC-H result, while 71 (0.31%) were HGSIL. Follow-up cervical dysplasia by colposcopic biopsy and cone biopsy was distributed proportionally between TIS and manual screening for both ASC-H and HGSIL categories. Cervical intraepithelial neoplasia (CIN II/III) was identified on follow-up biopsy of 41% TIS cases and 45% manually screened cases for ASC-H. In the HGSIL subset 71% of TIS cases and 69% manually screened cases showed CIN II/III on follow-up. TIS was 26% less sensitive relative to manual screening for ASC-H cases and 3% less sensitive for HGSIL. Conclusion: The similar rate of detection using TIS with an equal percentage of histologic correlation for ASC-H and HGSIL lesions on follow-up histology suggests patients screened by the TIS method are being sent for appropriate follow-up surveillance and treatment. A high-grade or possible high-grade lesion is as likely to be detected by TIS as by a manual screen. The similarities in relative sensitivity and specificity in a direct comparison between manual and TIS screening methodologies indicate that TIS compared to manual screening does not affect detection in patients with high-grade cervical lesions.
Objectives: Fine needle aspiration cytology (FNAC) has been employed in pre-operative diagnosis of salivary gland lesions for many years. Various studies in the existing literature have shown a wide range of sensitivity and diagnostic accuracy of cytologic diagnosis. This study was aimed at evaluating salivary gland FNAC for sensitivity, specificity and diagnostic accuracy at a tertiary care center. Materials and Methods: This study included 80 patients who underwent pre-operative FNAC followed by surgical procedure and histologic examination. The histologic diagnosis was considered as the gold standard. FNAC diagnosis was compared with the final histologic impression and concordance assessed. Sensitivity, specificity and diagnostic accuracy of FNAC for malignant lesions were calculated. Results: Of the 80 cases, majority (67.5%) involved the parotid gland. Eight cases (10%) were non-neoplastic lesions, comprised of sialadenitis, retention cyst and sialadenosis. Of a total of 72 neoplasms, 58 were benign and 14 were malignant salivary gland tumors. A cyto-histologic concordance of benign diagnosis was achieved in 85.7% of cases and for malignant lesions in 92.8% of the malignant tumors. FNAC showed a sensitivity of 92.8%, specificity of 93.9%, a positive predictive value of 81.2% and negative predictive value of 98.4% for malignant salivary gland tumors. There was one false-negative diagnosis and four false-positive cases diagnosed on FNAC. Conclusion: FNAC continues to be a reliable diagnostic technique in hands of an experienced cytopathologist. The sensitivity of diagnosis of malignant lesions is high, though the rate of tumor type-specific characterization is lower, due to variable cytomorphology. In difficult cases, histologic examination may be employed for accurate diagnosis.
Janie Roberson, Allison Wrenn, John Poole, Andrew Jaeger, Isam A Eltoum
CytoJournal 2013 10(1):3-3
Introduction: Constructing or renovating a laboratory can be both challenging and rewarding. UAB Cytology (UAB CY) recently undertook a project to relocate from a building constructed in 1928 to new space. UAB CY is part of an academic center that provides service to a large set of patients, support training of one cytotechnology program and one cytopathology fellowship training program and involve actively in research and scholarly activity. Our objectives were to provide a safe, aesthetically pleasing space and gain efficiencies through lean processes. Methods: The phases of any laboratory design project are Planning, Schematic Design (SD), Design Development (DD), Construction Documents (CD) and Construction. Lab personnel are most critical in the Planning phase. During this time stakeholders, relationships, budget, square footage and equipment were identified. Equipment lists, including what would be relocated, purchased new and projected for future growth ensure that utilities were matched to expected need. A chemical inventory was prepared and adequate storage space was planned. Regulatory and safety requirements were discussed. Tours and high level process flow diagrams helped architects and engineers understand the laboratory daily work. Future needs were addressed through a questionnaire which identified potential areas of growth and technological change. Throughout the project, decisions were driven by data from the planning phase. During the SD phase, objective information from the first phase was used by architects and planners to create a general floor plan. This was the basis of a series of meetings to brainstorm and suggest modifications. DD brings more detail to the plans with engineering, casework, equipment specifics, finishes. Design changes should be completed at this phase. The next phase, CD took the project from the lab purview into purely technical mode. Construction documents were used by the contractor for the bidding process and ultimately the Construction phase. Results: The project fitted out a total of 9,000 square feet; 4,000 laboratory and 5,000 office/support. Lab space includes areas for Prep, CT screening, sign out and Imaging. Adjacent space houses faculty offices and conferencing facilities. Transportation time was reduced (waste removal) by a Pneumatic Tube System, specimen drop window to Prep Lab and a pass thru window to the screening area. Open screening and prep areas allow visual management control. Efficiencies were gained by ergonomically placing CT Manual and Imaging microscopes and computers in close proximity, also facilitating a paperless workflow for additional savings. Logistically, closer proximity to Surgical Pathology maximized the natural synergies between the areas. Conclusions: Lab construction should be a systematic process based on sound principles for safety, high quality testing, and finance. Our detailed planning and design process can be a model for others undertaking similar projects
Transformation of epithelial cells by high-risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations often affecting tumor suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation profiles with genome-wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST, and TNFSF13) specific methylation in HPV-containing cell lines was confirmed by semi-quantitative methylation specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3, and PRDM14. Analysis of hrHPV-positive cervical scrapes revealed signifcantly increased methylation levels of the latter three genes in women with high-grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16 transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2-8, PHACTR3, and PRDM14 genres, in cervical carcinogenesis. These genes may provide promising triage markers to assess the presence of (pre)cancerous cervical lesions in hrHPV-positive women.
Oncogenic fusion genes that involve kinases have proven to be effective targets for therapy in a wide range of cancers. Unfortunately, the diagnostic approaches required to identify these events are struggling to keep pace with the diverse array of genetic alterations that occur in cancer. Diagnostic screening in solid tumours is particularly challenging, as many fusion genes occur with a low frequency. To overcome these limitations, we developed a capture enrichment strategy to enable high throughput transcript sequencing of the human kinome. This approach provides a global overview of kinase fusion events, irrespective of the identity of the fusion partner. To demonstrate the utility of this system we profiled one hundred non-small cell lung cancers and identified numerous genetic alterations impacting Fibroblast Growth Factor Receptor 3 (FGFR3) in lung squamous cell carcinoma and a novel ALK fusion partner in lung adenocarcinoma.
Recently mutations in the MED12 gene have been reported in 5.4% of prostate tumours from Caucasian patients analysed by exome sequencing (Barbieri et al., 2012). In >70% of prostate tumours with MED12 mutation, a recurrent p.L1224F mutation in exon 26 was found. In order to validate this MED12 p.L1224F mutation an unselected cohort of prostate tumours from Caucasian patients was analysed by Sanger sequencing. Overall, 223 prostate tumours and 3 lymph node metastases were analysed. The MED12 p.L1224F mutation could not be detected in any of the cases. So far, the recently reported MED12 p.L1224F mutation could not be validated in our unselected cohort of prostate tumours. Contrary to the findings of Barbieri and co-workers our data indicate either, that the p.L1224F mutation in the MED12 gene might play no role in prostate carcinogenesis, or this alteration might only be relevant in a small subgroup of tumours.
CD146 is an adhesion molecule localized at endothelial cell junctions and facilitates cell-cell interactions. The circulating soluble form (sCD146) lacks both the intracellular and the transmembrane domains. In this study we show that CD146 expression was significantly decreased in the lung tissue of smokers with chronic obstructive pulmonary disease (COPD) and also from rats exposed to second hand smoke (SHS). Concurrently, levels of sCD146 were increased in both the plasma and bronchoalveolar lavage fluid (BALF) of COPD patients as well as in BALF from rats exposed to SHS. Decreased or abolished CD146 protein expression in rat pulmonary micro- and macro-vascular endothelial cells was found after treatment with cigarette smoke extract (CSE), proinflammatory cytokine interleukin 18 (IL-18) or after silencing CD146 expression with siRNA. The decrease in CD146 protein was accompanied by increased endothelial monolayer permeability and enhanced macrophage infiltration in vitro. In CD146 knockout (KO) mice, distinct perivascular oedema was seen and increased numbers of inflammatory cells along with increased protein levels in BALF. Increased sCD146 was found in BALF and plasma from patients with COPD. The circulating plasma levels of sCD146 positively correlated with the presence of anti-endothelial cell antibodies (AECA). sCD146 in combination with AECA may be useful markers for early detection of COPD. Our study indicates that loss of CD146 function damages pulmonary endothelial integrity. This damage may represent a part of the pathophysiologic processes that are involved in the basic aetiology of COPD/emphysema.
Mucosal melanoma displays distinct clinical and epidemiological features compared to cutaneous melanoma. Here we used whole genome and whole exome sequencing to characterize the somatic alterations and mutation spectra in the genomes of ten mucosal melanomas. We observed somatic mutation rates that are considerably lower than occur in sun-exposed cutaneous melanoma, but comparable to the rates seen in cancers not associated with exposure to known mutagens. In particular, the mutation signatures are not indicative of ultraviolet light or tobacco smoke induced DNA damage. Genes previously reported as mutated in other cancers were also mutated in mucosal melanoma. Notably, there were substantially more copy number and structural variations in mucosal melanoma than have been reported in cutaneous melanoma. Thus, mucosal and cutaneous melanomas are distinct diseases with discrete genetic features. Our data suggest that different mechanisms underlie the genesis of these diseases and that structural variations play a more important role in mucosal than cutaneous melanomagenesis.
Background:
Recurrence patterns in patients who have undergone curative gastrectomy for advanced gastric carcinoma can be classified as peritoneal, hematogenous, or lymphatic. The aim of this study was to clarify differences in risk factors between these different types of recurrence pattern.
Methods:
Postoperative courses, including sites of recurrence and periods between surgery and recurrence, of patients who had undergone curative gastrectomy for advanced gastric carcinoma (more than pT2 invasion) were surveyed in detail. Clinicopathological factors were examined as potential independent risk factors for each recurrence pattern, based on recurrence-free survival, using multivariate analysis.
Results:
Multivariate analysis identified depth of tumor invasion (pT4 vs. pT2/3; hazard ratio (HR), 7.05; P < 0.001), number of lymph node metastases (pN2/3 vs. pN0/1; HR, 4.02; P = 0.001), and histological differentiation (G3/4 vs. G1/2; HR, 2.22; P = 0.041) as independent risk factors for peritoneal metastasis. The number of lymph node metastases (HR, 26.21; P < 0.001) and venous vessel invasion (HR, 5.09; P = 0.001) were identified as independent risk factors for hematogenous metastasis. The number of lymph node metastases (HR, 6.00; P = 0.007) and depth of tumor invasion (HR, 4.70; P = 0.023) were identified as independent risk factors for lymphatic metastasis.
Conclusions:
This study clarified differences in risk factors between various patterns of recurrence. Careful examination of risk factors could help prevent oversight of recurrences and improve detection of recurrences during follow-up. The number of lymph node metastases represents an independent risk factor for all three patterns of recurrence; thus, patients with multiple lymph node metastases warrant particular attention.
We report a rare case of lymphoepithelioma-like hepatocellular carcinoma. A 79-year-old Japanese man had undergone curative resection of extrahepatic bile ducts because of bile duct cancer 9 years prior. The bile duct cancer was diagnosed as mucosal adenocarcinoma, and the patient had been followed up every 6 months for the last 9 years. A recent computed tomography examination revealed a tumor, 4.2 cm in size, in the lateral segment of the liver. Based on the imaging findings, the tumor was diagnosed as hepatocellular carcinoma. Serology tests were negative for hepatitis B and C viruses. Chest and abdominal image analyses showed no evidence of metastasis, but a swollen lymph node was noted around the abdominal aorta. The patient subsequently underwent extended lateral segmentectomy and resection of the swollen lymph node. Microscopically, the tumor had the characteristic appearance of poorly differentiated hepatocellular carcinoma. Moreover, an abundant infiltration of inflammatory cells was observed in the tumor. Therefore, we diagnosed the tumor as lymphoepithelioma-like hepatocellular carcinoma. The resected para-aortic lymph node also had a carcinoma with features similar to those of the main tumor. The patient has been alive for 20 months since performance of the surgery. Since the first report of lymphoepithelioma-like hepatocellular carcinoma in 2000, only nine cases have been reported in the medical literature, and the clinicopathological features of the disease have not been well documented. Herein, we describe the clinicopathological features of this case for further understanding of the disease and review past cases in the literature.
Background:
Hepatocellular carcinoma (HCC) has a high predilection for portal vein invasion, and the prognosis of HCC with malignant portal vein invasion is extremely poor. The objective of this study was to investigate the outcomes and the prognostic factor of recurrence in HCC patients with malignant portal vein invasion.
Methods:
We retrospectively reviewed the clinicopathologic data and outcomes of 83 HCC patients with malignant portal vein invasion and 1,056 patients without portal vein invasion who underwent liver resection.
Results:
Increased serum alkaline phosphatase (ALP) levels, increased maximum tumor size, and intrahepatic metastasis were predisposing factors for malignant portal vein invasion by multivariate analysis. The median disease-free survival and overall survival of HCC patients with malignant portal vein invasion was 4.5 months and 25 months, respectively. The 1-year, 2-year, and 3-year disease-free survival rates were 30.6%, 26.1%, and 21.2%, respectively, and the overall survival rates for HCC patients with malignant portal vein invasion were 68.6%, 54.2%, and 41.6%, respectively. The initial detection site was the lung in HCC patients with portal vein invasion and the liver in HCC patients without portal vein invasion. C-reactive protein (CRP) was a significant independent predictor of tumor recurrence in HCC with malignant portal vein invasion after surgery.
Conclusions:
Increased ALP levels, increased maximum tumor size, and intrahepatic metastasis were independent predictors of malignant portal vein invasion in HCC. CRP level was closely associated with the predisposing factor of tumor recurrence in HCC patients with malignant portal vein invasion after a surgical resection, and lung metastasis was common.
Background:
Previous thyroid or parathyroid surgery induces scarring or distorts anatomy, and increases the risk of recurrent laryngeal nerve (RLN) injury for a reoperation. The benefit of intraoperative nerve monitoring (IONM) for re-exploration (a second nerve exploration) and reoperation has not been established.
Methods:
Two hundred and ten patients were given a thyroid or parathyroid reoperation at our hospital between 2001 and 2010. Using IONM, we re-explored 56 patients who had been operated on before June 2007. The injury rate in these patients was compared with that of the 15 patients re-explored without IONM between 2001 and 2006.
Results:
Of the 70 nerves that were re-explored using IONM, only one was incidentally injured, significantly fewer than the three injured in the 15 nerves re-explored without using IONM (1.43% vs. 20%, P = 0.0164).
Conclusions:
IONM helped prevent RLN damage when re-exploring nerves during thyroid and parathyroid surgery. We recommend the routine use of IONM in thyroid and parathyroid reoperations.
Background:
S100A7 signaling plays a critical role in the pathogenesis and progression of human breast cancers but the precise role and mechanism of S100A7 for tumor invasion remains unclear. in the present study, we investigated whether S100A7 overexpression could be mechanistically associated with the up-regulation of NF-κB, VEGF and MMP-9, resulting in the promotion of breast cancer cell invasion and growth, and vice versa.
Methods:
pcDNA3.1-S100A7 cDNA plasmid was constructed and transfected into the MDA-MB-468 cells. 4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect cell proliferation, Matrigel was used to detect cell mobility and invasion in vitro.The MMP-9 and VEGF expression and levels was detected by western blot and ELISA assay. NF-κB DNA binding activity was detected by Electrophoretic mobility shift assay.
Results:
Up-regulation of S100A7 by stable S100A7 cDNA transfection increased cell invasion and proliferation, whereas downregulation of S100A7 by small interfering RNA in S100A7 cDNA-transfected MDA-MB-468 cells decreased cell invasion and proliferation. Consistent with these results, we found that the up-regulation of S100A7 increased NF-κB DNA-binding activity and MMP-9 and VEGF expression. Down-regulation of S100A7 in S100A7 cDNA -transfected decreased NF-κB DNA-binding activity and MMP-9 and VEGF expression.
Conclusions:
Our data demonstrate that the S100A7 gene controls the proliferation and invasive potential of human MDA-MB-468 cells through regulation of NF-κB activity and its target genes, such as MMP-9 and VEGF expression. Down-regulation of S100A7 could be an effective approach for the down-regulation and inactivation of NF-κB and its target genes, such as MMP-9 and VEGF expression, resulting in the inhibition of invasion and growth.
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