Overexpression of Claudin Proteins in Esophageal Adenocarcinoma and Its Precursor Lesions (2010-09-06 01:36:59+01:00)

Claudins are components of tight junctions important in intercellular barriers and cell polarity. The authors identified upregulation of Claudins 3, 4, and 7 in gastric adenocarcinoma using Affymetrix U-133 oligonucleotide microarrays and immunohistochemistry (IHC). While normal gastric mucosa lacked Claudin 3, 4, and 7 expression, intestinal metaplasia and dysplasia showed these proteins. The authors hypothesized that Claudins would be similarly overexpressed in Barrett's esophagus (BE)/adenocarcinoma. Claudins 3, 4, and 7 gene expression was analyzed by Affymetrix U-133 microarrays in three esophageal adenocarcinomas, one case of BE, and three normal esophagi. IHC validation was performed using tissue microarrays constructed from esophageal resection specimens containing squamous (44 cases), gastric (40 cases), and nondysplastic BE (16 cases), low-grade and high-grade dysplasia (16 and 26 cases), adenocarcinoma (58 cases), and nodal metastases (27 cases). IHC staining was scored semiquantitatively (0+ to 4+). By microarray analysis, Claudin 3 showed a marked increase in mRNA expression compared with normal esophagus (approximately 100-fold). Claudins 4 and 7 were modestly increased (2.2- and 1.3-fold). By IHC, Claudin 3 expression was 1+ in most (>95%) normal squamous or gastric tissues and 2+ to 4+ in more than 80% of high-grade dysplasia, adenocarcinoma, and metastases specimens. Claudin 4 protein expression was 2+ or less in most squamous and gastric mucosa (>90%) but 3+ or 4+ in BE, low- and high-grade dysplasia, adenocarcinoma, and metastases specimens (>90%). Claudin 7 expression was minimal in squamous and gastric mucosa but strong (3+ to 4+) in BE and low-grade dysplasia. In high-grade dysplasia, adenocarcinoma, and metastases, Claudin 7 was less intense, with 60% to 70% staining 3+ or 4+ and 30% to 40% staining weakly (1+ or 2+). The findings suggest that alterations in Claudin proteins are an early event in tumorigenesis and may provide targets for diagnosis and directed therapy for esophageal adenocarcinoma and its precursors. (C) 2006 Lippincott Williams & Wilkins, Inc.

Coexpression of c-kit and bcl-2 in Small Cell Carcinoma and Large Cell Neuroendocrine Carcinoma of the Lung (2010-09-06 01:36:59+01:00)

It has been shown that tyrosine kinase oncoprotein c-kit and antiapoptotic molecule bcl-2 are overexpressed in several types of malignancy, including small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung. Whether these 2 molecules are coexpressed in lung neuroendocrine tumors has not been investigated. Here, we analyzed immunohistochemical results to determine expression and coexpression patterns of c-kit and bcl-2 in the spectrum of lung neuroendocrine tumors. Using a polyclonal antibody against c-kit and a monoclonal antibody against bcl-2, our data demonstrated that all 7 cases (100%) of SCLC included in this study were positive for both c-kit and bcl-2. Among 14 LCNECs, 7 (50%) stained positive for c-kit and 9 (64%) for bcl-2. All cases of high grade neuroendocrine carcinomas (SCLCs and LCNECs) that showed positive staining for c-kit coexpressed bcl-2. In contrast, all typical and atypical carcinoids (TC and AC) were negative for c-kit, and only 1 of 16 (6.3%) TCs and 1 of 6 (16.7%) ACs stained positive for bcl-2. These results indicate a progressive increase in the frequency of c-kit and bcl-2 expression and coexpression, from carcinoid tumors (TC and AC) to LCNEC and to SCLC. High grade neuroendocrine carcinomas are more likely to coexpress c-kit and bcl-2 when compared with carcinoid tumors. The high frequency of coexpression of these 2 molecules in high grade neuroendocrine carcinomas of the lung suggests that they may be involved in the carcinogenic pathway, given their important roles in carcinogenesis. Therapeutic targeting on both c-kit and bcl-2 molecules might be beneficial in the management of patients with high grade neuroendocrine carcinomas of the lung in the future. (C) 2007 Lippincott Williams & Wilkins, Inc.

Podoplanin (D2-40) is a Highly Effective Marker of Follicular Dendritic Cells (2010-09-05 13:15:27+01:00)

The monoclonal antibody D2-40 recognizes the membrane protein podoplanin, which is an established marker for germ cell tumors, mesotheliomas, and other tumor types and is also expressed in a variety of normal cells including follicular dendritic cells (FDCs). To determine whether podoplanin represents an effective FDC marker for pathologic lymph nodes, we compared immunohistochemical studies (sensitivity, staining patterns, and intensity of staining) for podoplanin (D2-40) with those of the traditional FDC markers CD21, CD35, and clusterin. Paraffin sections of 26 lymph nodes were analyzed, including 4 cases of nodular lymphocyte-predominant Hodgkin lymphoma, 4 angioimmunoblastic T-cell lymphoma, 8 follicular lymphoma (including 3 cases with a component of diffuse large B-cell lymphoma), 5 hyaline-vascular Castleman disease, and 5 reactive lymph nodes with follicular hyperplasia. In all cases, qualitatively and quantitatively podoplanin represented a highly effective marker for detection of FDCs, with staining intensity equal to or greater than that observed for other FDC markers. This study demonstrates that podoplanin is an excellent marker for FDCs and adds to its growing list of diagnostic applications. (C) 2009 Lippincott Williams & Wilkins, Inc.

Lack of Immunohistochemical Detection of VEGF in Prostate Carcinoma (2010-09-04 15:13:08+01:00)

Background: Vascular endothelial growth factor (VEGF) has been implicated in tumor angiogenesis and is a potential therapeutic target in prostatic adenocarcinoma (PrCa). Immunohistochemical (IHC) analysis has been used to demonstrate VEGF expression in PrCa, and in various other tumors including breast carcinoma, renal cell carcinoma, hepatocellular carcinoma, and gliomas. Prior studies have reported markedly varied VEGF expression in benign prostatic hyperplasia (0% to 100%) and PrCa (40% to 100%). The objective of this study was to measure VEGF expression in PrCa specimens, using IHC analysis with antibodies from different manufacturers and different antigen retrieval techniques. Design: Cases were identified from an ongoing study analyzing 1270 cases of PrCa diagnosed at various Veterans Health Administration hospitals in the New England region during 1991 to 1995. From this study population, 50 cases (including 40 biopsies, 8 transurethral resections of prostate, and 2 radical resections) were selected. In all cases, tissues were fixed in 10% formalin and embedded in paraffin. Four different antibodies were used for IHC using indirect peroxidase method. For each antibody, different dilutions and antigen retrieval methods (steam with ethylene diamine tetra-acetate, steam with low pH, water bath with target unmasking fluid, trypsin, proteinase K) were tested. EnVision+ system was used to overcome nonspecific staining. Appropriate positive and negative controls were used. Results: Using different antibodies, positive staining of varying intensity was seen in benign glands, malignant glands, endothelial cells, and fibromuscular stroma. Some cases showed cytoplasmic and granular staining in prostatic glands. However, the staining disappeared in all cases when EnVision+ system was used to block nonspecific staining except for focal and minimal staining in the endothelial cells. Conclusions: Our results show that when nonspecific staining is blocked, no staining is found for VEGF within the prostate, in either benign or malignant glands. The reasons for the granular and nonspecific staining are unclear at present. Our study may help to explain variable results reported in previous studies, and suggests caution in interpreting VEGF expression in studies of PrCa and benign glands. (C) 2009 Lippincott Williams & Wilkins, Inc.

TTF-1 Positivity in Metastatic Colorectal Carcinoma: A Diagnostic Pitfall in Transbronchial Biopsy (2010-09-01 10:33:26+01:00)

No abstract available

TTF-1 Positivity in Metastatic Colorectal Carcinoma: A Diagnostic Pitfall in Transbronchial Biopsy (2010-08-26 13:52:29+01:00)

No abstract available

Relationship Between p73 Polymorphism and the Immunohistochemical Profile of the Full-length (TAp73) and NH2-truncated ([DELTA]Np73) Isoforms in Tunisian Patients (2010-08-26 13:52:29+01:00)

Background: We examined the association of one linked GC/AT polymorphism at p73 with the risk of colorectal cancer. Aim: In this study, we investigated whether this polymorphism was related to the risk of colorectal cancer, and whether there were relationships between the polymorphism and loss of heterozygosity, protein expression, or clinicopathologic variables. Materials and Methods: The p73 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 150 Tunisian patients with colorectal cancer and in 204 healthy control subjects. Immunohistochemistry was performed on normal mucosa, primary tumor, and metastasis. Results: The frequencies of the genotypes were 52% for wild-type (GC/GC), 31% for heterozygotes (GC/AT), and 17% for variants (AT/AT) in patients, and 54%, 35%, and 11% in controls, respectively. There were no significant differences of the frequencies of the 3 genotypes between the patients and controls (P=0.11). We did not find any relationship of the genotypes with clinicopathologic features of patients. We found that patients with the GC/GC genotype had a significantly more favorable clinical outcome than the patients with the AT variants (AT/AT or GC/AT genotype). There were no significant difference between tumoral immunostaining and p73 polymorphism (P=0.16) but we found that the samples carried the AT allele showed a tendency to be more stained in tumor. No loss of heterozygosity was observed at p73 locus. Our results suggest that the AT/AT genotype is significantly associated with poor prognosis in colorectal cancer. All these findings suggest that p73 polymorphism analysis may provide useful prognostic information for colorectal cancer patients. (C) 2010 Lippincott Williams & Wilkins, Inc.

Nuclear Survivin Is Associated With Malignant Potential in Epithelial Ovarian Carcinoma (2010-08-20 01:20:15+01:00)

Survivin is a unique member of the inhibitor of apoptosis (IAP) protein family. As abnormal inhibition of apoptosis during homeostasis is considered a critical step in the initiation of cancer, we investigated prognostic value of survivin expression in epithelial ovarian carcinomas. We carried out immunohistochemical experiments using a polyclonal antisurvivin antibody to stain formalin-fixed paraffin-embedded sections from 91 patients with ovarian tumors, including 10 cystadenomas, 17 borderline tumors, and 64 epithelial ovarian carcinomas. Nuclear and cytoplasmic survivin staining was scored separately. Survivin expression was undetectable in ovarian cystadenomas and was weakly detected in 4 of 17 (23.53%) borderline tumors. In contrast, nuclear and cytoplasmic survivin staining was observed in 55 of 64 (85.94%) epithelial ovarian carcinomas. Scoring on the basis of the percentage of survivin nuclear-positive cells indicated that nuclear survivin expression was associated significantly with clinical stage, histologic grade, proliferating cell nuclear antigen (PCNA)-labeling index, and clinical outcome in ovarian epithelial carcinoma patients (P<0.01). Taken together, the results of this study provide evidence that nuclear survivin expression is a strong, independent prognostic marker for poor clinical outcomes in epithelial ovarian carcinoma. (C) 2010 Lippincott Williams & Wilkins, Inc.

Development and Characterization of a JAK-2-specific Antibody Suitable for Immunohistochemical Investigative Studies (2010-08-20 01:20:15+01:00)

Janus kinases (JAKs) are tyrosine kinases called JAK-1, JAK-2, JAK-3, and Tyk-2, which have been shown to participate in the signaling pathways of several cytokines that are believed to play a key role in several autoimmune-mediated disorders including rheumatoid arthritis (RA). However, the availability of JAK-specific antibodies to be used in investigative efficacy studies in RA models is very limited. Therefore, in this study we developed and characterized a JAK-2-specific antibody that was used to evaluate its immunohistochemical expression in the joints of a rat adjuvant-induced arthritis (rAIA) RA preclinical model. An immunogen peptide sequence design was used to generate JAK-2-specific mouse, rat, and human polyclonal antibodies. JAK-2 plasmid cDNA was then generated and HEK293 transfected cell lines, gel electrophoresis, immunoblotting, and immunohistochemistry were used to further characterize the generated JAK-2 antibodies. We show that the generated JAK-2 antibody exhibits specificity and lacks cross-reactivity to JAK-1 and JAK-3. In addition, marked JAK-2 expression is shown in the rAIA in mixed inflammatory cells (macrophages and neutrophils), mast cells, and bone marrow elements. In conclusion, we show the development and characterization of a JAK-2-specific antibody that can be used in investigative and mechanistic studies such as preclinical efficacy models. (C) 2010 Lippincott Williams & Wilkins, Inc.

Expression of 6 Common Antigenic Markers in Invasive Ductal Breast Carcinoma: Potential Clinical Implications (2010-08-20 01:20:15+01:00)

Expression of estrogen (ER) and progesterone receptors, c-erbB-2 oncogene, mutant p53 antioncogene (mp53), e-cadherin adhesion, and apoptotic caspase-8 antigens in tumor relative to matched normal tissue specimens from 102 unselected patients with primary ductal breast carcinoma of various tumor grades was assessed by immunohistochemistry and correlated with patient's biologic and clinical features, such as age, menstrual status, age of menarche, tumor grade and diameter, the presence or absence of metastases, and number of infiltrated lymph nodes. We observed association of e-cadherin adhesion, ER and progesterone antigen marker expression with low histologic grade tumors and limited number of lymph node metastases and of c-erbB-2, mp53, and casp-8 antigen marker expression with high histologic grade tumors and increased number of lymph node metastases. We also observed strong correlation (P<0.05) between 4 of the 6 biomarkers and 4 of the 7 patient/tumor parameters examined. Our findings support the hypothesis of independent expression of these 4 strong biomarkers and reveal that nearly 40% of all breast tumor cases studied express similar proportions of 2 major phenotypic combinations [ER/c-erbB-2/mp53/casp-8: +/+/-/+ (19.6%) & +/-/-/+ (17.8%)]. We conclude that, in agreement with earlier reports, our findings support the diagnostic and potential prognostic value of these markers in the clinical assessment of breast cancer. (C) 2010 Lippincott Williams & Wilkins, Inc.

Protocol for PTEN Expression by Immunohistochemistry in Formalin-fixed Paraffin-embedded Human Breast Carcinoma (2010-06-25 00:51:15+01:00)

The PI3K/PTEN pathway plays a major role in carcinogenesis. Dysregulation of this pathway occurs frequently in breast cancer, and loss of PTEN expression is emerging as a potentially important mechanism of resistance to the widely used anti-HER2 therapy, trastuzumab. However, assays for loss of PTEN expression have suffered from lack of consistency. Here, we describe an automated and reliable protocol for PTEN protein expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue that can be easily incorporated into clinical trials. (C) 2010 Lippincott Williams & Wilkins, Inc.

Increased Detection of Metastatic Melanoma in Pediatric Sentinel Lymph Node Biopsies Using RT-PCR on Paraffin-embedded Tissue (2010-06-25 00:51:15+01:00)

Sentinel lymph node (SLN) biopsy has become integral in the staging of patients with melanoma, and entails detailed histologic examination with immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase transcripts has been used to increase sensitivity but requires a dedicated piece of tissue that does not undergo histologic examination. We developed a nested RT-PCR assay for tyrosinase applicable on paraffin-embedded tissue and applied this to a series of SLNs from pediatric patients with melanoma. Thirty-six SLNs from 4 females and 4 males were included in the study. Eight lymph nodes with reactive changes were included as controls. SLNs were examined histologically and immunohistochemically for S100, tyrosinase, and MART1. Seven patients had between 1 and 4 morphologically-positive SLNs and one patient had negative SLNs (HISTO+; 12/36, 33%). Three lymph nodes were excluded from molecular analysis owing to inadequate RNA, and 29 of the remaining 33 nodes were positive (MOL+; 88%). All patients had at least 1 SLN positive by RT-PCR. Twelve were HISTO+/MOL+; 17 were HISTO-/MOL+; and 4 were HISTO-/MOL-. All control lymph nodes were negative for tyrosinase transcripts. The application of RT-PCR for tyrosinase to paraffin-embedded tissue significantly increased the number of positive SLNs and upstaged one patient from negative to positive. The prognostic implications of such findings require further investigation, especially in the pediatric age group. Nonetheless, this technique provides a useful tool to determine the clinical significance of RT-PCR positivity in melanoma SLNs. (C) 2010 Lippincott Williams & Wilkins, Inc.

Double Immunohistochemical Staining Method for HIF-1[alpha] and its Regulators PHD2 and PHD3 in Formalin-fixed Paraffin-embedded Tissues (2010-06-25 00:51:15+01:00)

Hypoxia-inducible factor (HIF-1[alpha]) is expressed in the nuclei of tumor cells under hypoxic conditions, and is regulated, in part, by cytoplasmic prolyl hydroxylases (PHDs). As HIF-1[alpha] is selectively expressed in tumor cells, inhibitors are being developed for cancer therapy. Although methods for the detection of HIF-1[alpha] and PHDs are available, an immunohistochemical double staining method for these markers in individual tumor cells is not available. For method development a human squamous cell carcinoma (SCC) xenograft, A253, was used as a known positive control tissue for HIF-1[alpha] in well-differentiated areas without microvessels. This laboratory showed that tumor cells in these areas are strongly positive for hypoxia markers. Another human, poorly differentiated SCC xenograft, FaDu, without hypoxic areas, was used as a negative control. PHD2 and 3 immunostaining was optimized individually using the human kidney. To optimize HIF-1[alpha] detection the pressure cooker time for antigen retrieval, concentration of the primary antibody, amplification reagent, and DAB development time were decreased. Casein blocking further decreased the background. Double staining resulted in brown nuclei for HIF-1[alpha] (DAB), and pink cytoplasmic staining for PHD2, 3 (fast red). The isotype-matched controls were negative. Normal human tissues had no detectable HIF-1[alpha], but expressed PHD2, 3. The potential use of this new and improved method was confirmed by analyzing 15 surgical biopsies of oropharyngeal SCC of which 6 were positive for HIF-1[alpha]. This new method defined the optimal conditions for detection of HIF-1[alpha] and PHDs in individual tumor cells and could have a diagnostic and therapeutic potential. (C) 2010 Lippincott Williams & Wilkins, Inc.

About a Case of Diffuse Endometrial Squamous Metaplasia After Resectoscopic Myomectomy: A Potential Diagnostic Pitfall for Gynecologists and Pathologists (2010-06-25 00:51:15+01:00)

We report a case of diffuse endometrial squamous metaplasia found after in a resectoscopic myomectomy specimen. A 35-year-old woman underwent an office hysteroscopy that showed a submucosal leiomyoma. After pharmacologic treatment with a GnRH gonadotropin-releasing hormone agonists (GnRHa) leuprolide acetate, the patient underwent a resectoscopic myomectomy. Histologic examination showed fragments of myometrial tissue with the foci of endometrial glands with diffuse features of squamous metaplasia; in part classical, mature type; and in part immature type, and the so-called "morula type." Presence of endometrial squamous metaplasia in the endometrium may produce dramatic histologic changes on biopsies and sometimes it may be difficult to distinguish it from primitive primary carcinomas of nonendometrioid histology, representing therefore, a potential diagnostic pitfall. GnRHa therapy could play a possible role in the onset of squamous metaplasia. So the pathologist and gynecologist must take into account this possible metaplastic change for a correct clinicopathologic assessment and to avoid overtreatment. (C) 2010 Lippincott Williams & Wilkins, Inc.

MUM1/IRF4: A Review (2010-06-25 00:51:15+01:00)

MUM1/IRF4 protein is a member of the interferon regulatory factor (IRF) family of transcriptional factors initially described as downstream regulators of interferon signaling. The quantity of this factor varies within the hematopoietic system in a lineage and stage-specific way. It is considered to be a key regulator of several steps in lymphoid, myeloid, and dendritic cell differentiation and maturation. MUM1/IRF4 expression is observed in many lymphoid and myeloid malignancies, and may be a promising target for the treatment of some of these neoplasms. We reviewed the literature on MUM1/IRF4, with emphasis on the pathologic aspects of this marker in reactive and malignant hematologic and nonhematologic conditions. (C) 2010 Lippincott Williams & Wilkins, Inc.

CEMOVIS on a pathogen: analysis of Bacillus anthracis spores (2010-08-26T13:47:33Z)

Background information: Under conditions of starvation bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the etiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analyzing the organization of the spore and the changes during germination at the electron microscopy level have been only done with techniques relying on fixation with aldehydes and osmium and subsequent deshydration, which can produce artefacts. Results and conclusions: In this study we describe the morphology of dormant spores using cryoelectron microscopy of vitrified sections (CEMOVIS). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore we chose to analyse spores of the closely related non pathogen B. cereus T. For none of the investigated structures we could find a difference in morphology induced by aldehyde fixation compared to the native preparations for CEMOVIS. This result legitimises to work with aldehyde fixed spores from B. anthracis. Using CEMOVIS we describe here two new structures present in the spore: A rectangular structure, which connects the BclA filaments with the basal layer of the exosporium and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated and the layered organization of the coat as well as the repetitive structure within it remain unchanged.

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells (2010-08-24T15:23:15Z)

Background information. The Golgi apparatus (GA) has an essential role in membrane traffic determining the assembly and delivery of uroplakins to the apical plasma membrane (APM) of superficial uroepithelial cells (UCs) of urinary bladder. Uroplakins are synchronously and uniformly delivered from the GA to the APM by discoidal fusiform-shaped vesicles (DFVs), however the mechanism of uroplakin delivery is not known. We have used the culture model of UCs with the capacity to undergo the terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. Results. By three-dimensional localisation using confocal microscopy of immunofluorescence labelled GA-related markers (GM130, GS15, GS28, giantin), uroepithelial differentiation-related markers (uroplakins), microtubules (MTs; α-tubulin) and intermediate filaments (CK7, CK20), we found that in non-differentiated, uroplakin-negative UCs the GA is mostly organized as the single ribbon-like structure close to the nucleus, while in differentiated, uroplakin-positive UCs the GA is fragmented and spread almost through the entire cell. The fluorescence recovery after photobleaching experiments on the UCs transfected with β1,4-galactosyl transferase (GalT) fused to fluorescent protein showed that Golgi-resident enzyme cycles freely within ribbon-like GA but not within fragmented GA. By correlative light and electron microscopy we examined the GA fragments in cells expressing uroplakins. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. Furthermore, we demonstrated that the reorganization of GA into a fragmented form is associated with the impairment of the MT organization in the basal, central and subapical cytoplasm and accumulation of intermediate filaments in the apical cytoplasm that could affect kinetics of MT star leading to the peripheral fragmentation of the GA in differentiated UCs. Conclusions. The fragmentation of the GA and its spreading to the cell periphery is one of the key events, which promote the uniform delivery of uroplakins over the entire APM of differentiating UCs and thus have a major importance in the final proper formation and maintenance of the blood-urine barrier.

Spatial organization of the transforming MHC class II compartment (2010-08-16T13:09:45Z)

Background information: Dendritic cells (DC) continuously capture pathogens and process them into small peptides within the endolysosomal compartment, the MIIC (major histocompatibility complex class II containing compartment). In MIICs peptides are loaded onto MHC class II and rapidly redistributed to the cell surface. This redistribution is accompanied by profound changes of the MIICs into tubular structures. An emerging concept is that MIIC tubulation provides a way to transport MHC class II-peptide complexes to the cell surface, either directly or through vesicular intermediates. To obtain spatial information on the reorganization of the MIICs during DC maturation, we performed electron tomography on cryo-immobilized and freeze-substituted mouse DCs after stimulation with lipopolysaccharide (LPS). Results: In non-stimulated DCs, MIICs are mostly spherical. After 3h LPS stimulation, individual MIICs transform into tubular structures. 3D reconstruction showed that the MIICs frequently display fusion profiles and upon 6h LPS stimulation, MIICs become more interconnected, thereby creating large MIIC reticula. Microtubules and microfilaments align these MIICs and reveal physical connections. In our tomograms we also identified a separate population of MIIC-like intermediates particularly at extended ends of MIIC tubules and in close proximity to the trans golgi network. No fusion events were captured between reticular MIICs and the plasma membrane. Conclusions: Our data indicate that MIICs have the capacity to fuse together, whereby the cytoskeleton possibly provides a scaffold for the MIIC shape change and directionality. MIIC-like intermediates may represent MHC class II carriers.

Inhibition of GSK3{beta} enhances both adhesive and signaling activities of {beta}-catenin in mouse embryonic stem cells (2010-07-14T08:38:23Z)

Background.GSK3β kinase regulates the expression level and activity of various target proteins,including β-catenin. β-catenin is a co-activator of Wnt-dependent genes as well as a partner for trans-membrane cadherins to mediate cell-to-cell adhesion. In some cases, inhibition of GSK3β activity was shown to promote self-renewal of embryonic stem cells, but immediate effects of GSK3β inhibitors in these cells still remain elusive. Results.Here we address the effects of GSK3β inhibitors 6-Bromoindirubin-3′-oxime and CHIR99021 in mouse embryonic stem cells (mESCs), focusing on modulation of β-catenin activities. We found that upon GSK3β inhibition the colonies of undifferentiated mouse embryonic stem cells acquire more compact morphology. This change is paralleled by two somewhat polar effects: (1) the accumulation of β-catenin, which is co-localized with E-cadherin at the plasma membrane and the cytoplasmic, tyrosine unphosphorylated β-catenin, which is able to bind the GST-fused cytoplasmic domain of E-cadherin; (2) the accumulation of the tyrosine phosphorylated β-catenin and its nuclear translocation that is accompanied by activation of the Tcf/β-catenin-dependent transcription of Top-Flash reporter. The Tcf-mediated activation, however, does not affect the majority of analyzed Wnt-responsive genes involved in epithelial-mesenchymal transition or cell cycle progression, suggesting that the adhesive function of β-catenin is more operative than transcriptional in undifferentiated mESCs. Treatment with BIO was shown to decrease proliferation rates of mESCs. This is not due to apoptosis, but rather to accumulation of cells in G1 phase of the cell cycle and is accompanied by down-regulation of the c-myc mRNA content. Conclusion.Our data suggest that inhibition of GSK3β activity in mESCs enhances both the β-catenin/E-cadherin mediated adhesion and the Tcf/β-catenin-dependent transcription, but does not activate transcription in majority of the examined genes involved in epithelial-mesenchymal transition and cell cycle progression.

Tissue-type plasminogen activator induces plasmin-dependent proteolysis of intracellular neuronal nitric oxide synthase (2010-07-16T16:00:58Z)

Background information: Despite its pro-fibrinolytic activity, tissue-type plasminogen activator (tPA) is a serine protease known to influence a number of physiological and pathological functions in the central nervous system. Accordingly, tPA was reported to mediate some of its functions in the central nervous system through either N-methyl-D-aspartate receptors, Low-density lipoprotein-related receptor protein or annexin-II. Results: We provide here both in vitro and in vivo evidence that tPA could mediate proteolysis and subsequent delocalization of neuronal nitric oxide synthase , thereby reducing endogenous neuronal nitric oxide release. We also demonstrate that although this effect is independent of N-methyl-D-aspartate receptors, Low-density lipoprotein-related receptor protein signaling and calpain-mediated proteolysis, it is dependent on the ability of tPA to promote conversion of plasminogen into plasmin. Conclusion: Altogether, these data provide a new function of tPA in the central nervous system, which likely contributes to its pleiotropic functions.

Tissue-type plasminogen activator induces plasmin-dependent proteolysis of intracellular neuronal nitric oxide synthase (2010-08-18)

Background information. Despite its pro-fibrinolytic activity, tPA (tissue plasminogen activator) is a serine protease known to influence a number of physiological and pathological functions in the central nervous system. Accordingly, tPA was reported to mediate some of its functions in the central nervous system through NMDA (N-methyl-D-aspartate) receptors, LRP (low-density lipoprotein receptor-related protein) or annexin II.

Results. We provide here both in vitro and in vivo evidence that tPA could mediate proteolysis and subsequent delocalization of neuronal nitric oxide synthase, thereby reducing endogenous neuronal nitric oxide release. We also demonstrate that although this effect is independent of NMDA receptors, LRP signalling and calpain-mediated proteolysis, it is dependent on the ability of tPA to promote the conversion of plasminogen into plasmin.

Conclusion. Altogether, these results demonstrate a new function for tPA in the central nervous system, which most likely contributes to its pleiotropic functions.

Bimolecular fluorescence complementation analysis of eukaryotic fusion products (2010-08-06)

Background information. Cell fusion is known to underlie key developmental processes in humans and is postulated to contribute to tissue maintenance and even carcinogenesis. The mechanistic details of cell fusion, especially between different cell types, have been difficult to characterize because of the dynamic nature of the process and inadequate means to track fusion products over time. Here we introduce an inducible system for detecting and tracking live cell fusion products in vitro and potentially in vivo. This system is based on BiFC (bimolecular fluorescence complementation) analysis. In this approach, two proteins that can interact with each other are joined to fragments of a fluorescent protein and are expressed in separate cells. The interaction of said proteins after cell fusion produces a fluorescent signal, enabling the identification and tracking of fusion products over time.

Results. Long-term tracking of fused p53-deficient cells revealed that hybrid cells were capable of proliferation. In some cases, proliferation was preceded by nuclear fusion and division was asymmetric (69%±2% of proliferating hybrids), suggesting chromosomal instability. In addition, asymmetric division following proliferation could give rise to progeny indistinguishable from unfused counterparts.

Conclusions. These results support the possibility that the chromosomal instability characteristic of tumour cells may be incurred as a consequence of cell fusion and suggest that the role of cell fusion in carcinogenesis may have been masked to this point for lack of an inducible method to track cell fusion. In sum, the BiFC-based approach described here allows for comprehensive studies of the mechanism and biological impact of cell fusion in nature.

Tackling the pathogenesis of RNA nuclear retention in myotonic dystrophy (2010-07-23)

DM1 (myotonic dystrophy type I) is a common form of muscular dystrophy that affects mainly adults. It is a disease that belongs to the group of defective RNA export diseases, since a major part of the pathogenic mechanism of the disease is the retention of the mutant transcripts in the cell nucleus. The presence of an expanded CUG trinucleotide repeat in the 3′-UTR (3′-untranslated region) of the DMPK (myotonic dystrophy protein kinase) gene causes the attraction of RNA-binding proteins by the nuclear-located mutant transcripts. As a result of the occupation of the RNA-binding proteins, there is defective mis-splicing of several cellular transcripts. This is believed to be a major pathogenic mechanism of the disease and any attempt to repair the activities of the RNA-binding proteins or target the mutant transcripts should be beneficial for the patients. Certain approaches have been described in the literature and they demonstrate progress in various directions. The purpose of the present review is to summarize the successful attempts to tackle the pathogenesis caused by nuclear retention of mutant transcripts in myotonic dystrophy and to discuss the possible gains from such approaches.

Transcriptional control of genes involved in ciliogenesis: a first step in making cilia (2010-07-09)

Cilia and flagella have essential functions in a wide range of organisms. Cilia assembly is dynamic during development and different types of cilia are found in multicellular organisms. How this dynamic and specific assembly is regulated remains an important question in cilia biology. In metazoans, the regulation of the overall expression level of key components necessary for cilia assembly or function is an important way to achieve ciliogenesis control. The FOXJ1 (forkhead box J1) and RFX (regulatory factor X) family of transcription factors have been shown to be important players in controlling ciliary gene expression. They fulfill a complementary and synergistic function by regulating specific and common target genes. FOXJ1 is essential to allow for the assembly of motile cilia in vertebrates through the regulation of genes specific to motile cilia or necessary for basal body apical transport, whereas RFX proteins are necessary to assemble both primary and motile cilia in metazoans, in particular, by regulating genes involved in intraflagellar transport. Recently, different transcription factors playing specific roles in cilia biogenesis and physiology have also been discovered. All these factors are subject to complex regulation to allow for the dynamic and specific regulation of ciliogenesis in metazoans.

Sodium fluoride induces podosome formation in endothelial cells (2010-07-06)

Background information. Fluoride is a well-known G-protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin-based structures endowed with adhesion and matrix-degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells.

Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell-division cycle 42), Rac1 and RhoA activities are stimulated in NaF-treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF-induced podosomes are fully competent for matrix protein degradation.

Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.

Behavior of a viscoelastic valveless pump: a simple theory with experimental validation (2010-08-31T00:00:00Z)

Background: A valveless pump generates a unidirectional net flow of fluid around a closed loop of soft viscoelastic tubing that is rhythmically compressed at one point. The tubing must have at least two sections with two different stiffnesses. When a short segment of the tube is squeezed asymmetrically at certain frequencies, net flow of fluid around the loop can occur without valves. Methods: Partial differential equations for the pressures, volumes, and flows define a simple one-dimensional model of such a pump, based upon elementary physical principles. Numerical computations on a personal computer can predict measured net flows. Results: Net flow varies with the frequency and waveform of compression used to excite the pump, as well as with the site of compression and the stiffness and viscosity of the tubing. Net flows on the order of 1 ml/sec are obtained in a water-filled loop including 46 cm of stiffer plastic (Tygon) laboratory tubing and 70 cm of softer latex rubber tubing. Conclusions: The heretofore mysterious phenomenon of valveless pumping can be described in terms of classical Newtonian physics, in which viscous damping in the walls of the pump is included. Studying valveless pumps in the laboratory and modeling their behavior numerically provides a low-cost, engaging, and instructive exercise for research and teaching in biomedical engineering.

Sequential algorithm for life threatening cardiac pathologies detection based on mean signal strength and EMD functions (2010-09-04T00:00:00Z)

Background: Ventricular tachycardia (VT) and ventricular fibrillation (VF) are the most serious cardiac arrhythmias that require quick and accurate detection to save lives. Automated external defibrillators (AEDs) have been developed to recognize these severe cardiac arrhythmias using complex algorithms inside it and determine if an electric shock should in fact be delivered to reset the cardiac rhythm and restore spontaneous circulation. Improving AED safety and efficacy by devising new algorithms which can more accurately distinguish shockable from non-shockable rhythms is a requirement of the present-day because of their uses in public places.MethodIn this paper, we propose a sequential detection algorithm to separate these severe cardiac pathologies from other arrhythmias based on the mean absolute value of the signal, certain low-order intrinsic mode functions (IMFs) of the Empirical Mode Decomposition (EMD) analysis of the signal and a heart rate determination technique. First, we propose a direct waveform quantification based approach to separate VT plus VF from other arrhythmias. The quantification of the electrocardiographic waveforms is made by calculating the mean absolute value of the signal, called the mean signal strength. Then we use the IMFs, which have higher degree of similarity with the VF in comparison to VT, to separate VF from VTVF signals. At the last stage, a simple rate determination technique is used to calculate the heart rate of VT signals and the amplitude of the VF signals is measured to separate the coarse VF from VF. After these three stages of sequential detection procedure, we recognize the two components of shockable rhythms separately. Results: The efficacy of the proposed algorithm has been verified and compared with other existing algorithms, e.g., HILB [1], PSR [2], SPEC [3], TCI [4], Count [5], using the MIT-BIH Arrhythmia Database, Creighton University Ventricular Tachyarrhythmia Database and MIT-BIH Malignant Ventricular Arrhythmia Database. Four quality parameters (e.g., sensitivity, specificity, positive predictivity, and accuracy) were calculated to ascertain the quality of the proposed and other comparing algorithms. Comparative results have been presented on the identification of VTVF, VF and shockable rhythms (VF + VT above 180 bpm). Conclusions: The results show significantly improved performance of the proposed EMD-based novel method as compared to other reported techniques in detecting the life threatening cardiac arrhythmias from a set of large databases.

Classification of videocapsule endoscopy image patterns: comparative analysis between patients with celiac disease and normal individuals (2010-09-04T00:00:00Z)

Background: Quantitative disease markers were developed to assess videocapsule images acquired from celiac disease patients with villous atrophy, and from control patients.MethodCapsule endoscopy videoclip images (576x576 pixels) were acquired at 2/second frame rate (11 celiacs, 10 controls) at regions: 1. bulb, 2. duodenum, 3. jejunum, 4. ileum and 5. distal ileum. Each of 200 images per videoclip (=100s) were subdivided into 10x10 pixel subimages for which mean grayscale brightness level and its standard deviation (texture) were calculated. Pooled subimage values were grouped into low, intermediate, and high texture bands, and mean brightness, texture, and number of subimages in each band (nine features in all) were used for quantifying regions 1-5, and to determine the three best features for threshold and incremental learning classification. Classifiers were developed using 6 celiac and 5 control patients' data as exemplars, and tested on 5 celiacs and 5 controls. Results: Pooled from all regions, the threshold classifier had 80% sensitivity and 96% specificity and the incremental classifier had 88% sensitivity and 80% specificity for predicting celiac versus control videoclips in the test set. Trends of increasing texture from regions 1 to 5 occurred in the low and high texture bands in celiacs, and the number of subimages in the low texture band diminished (r2>0.5). No trends occurred in controls. Conclusions: Celiac videocapsule images have textural properties that vary linearly along the small intestine. Quantitative markers can assist in screening for celiac disease and localize extent and degree of pathology throughout the small intestine.

Surface EMG pattern recognition for real-time control of a wrist exoskeleton (2010-08-26T00:00:00Z)

Background: Surface electromyography (sEMG) signals have been used in numerous studies for the classification of hand gestures and movements and successfully implemented in the position control of different prosthetic hands for amputees. sEMG could also potentially be used for controlling wearable devices which could assist persons with reduced muscle mass, such as those suffering from sarcopenia. While using sEMG for position control, estimation of the intended torque of the user could also provide sufficient information for an effective force control of the hand prosthesis or assistive device. This paper presents the use of pattern recognition to estimate the torque applied by a human wrist and its real-time implementation to control a novel two degree of freedom wrist exoskeleton prototype (WEP), which was specifically developed for this work.MethodBoth sEMG data from four muscles of the forearm and wrist torque were collected from eight volunteers by using a custom-made testing rig. The features that were extracted from the sEMG signals included root mean square (rms) EMG amplitude, autoregressive (AR) model coefficients and waveform length. Support Vector Machines (SVM) was employed to extract classes of different force intensity from the sEMG signals. After assessing the off-line performance of the used classification technique, the WEP was used to validate in real-time the proposed classification scheme. Results: The data gathered from the volunteers were divided into two sets, one with nineteen classes and the second with thirteen classes. Each set of data was further divided into training and testing data. It was observed that the average testing accuracy in the case of nineteen classes was about 88% whereas the average accuracy in the case of thirteen classes reached about 96%. Classification and control algorithm implemented in the WEP was executed in less than 125 ms. Conclusions: The results of this study showed that classification of EMG signals by separating different levels of torque is possible for wrist motion and the use of only four EMG channels is suitable. The study also showed that SVM classification technique is suitable for real-time classification of sEMG signals and can be effectively implemented for controlling an exoskeleton device for assisting the wrist.

Automatic segmentation of coronary angiograms based on fuzzy inferring and probabilistic tracking (2010-08-20T00:00:00Z)

Background: Segmentation of the coronary angiogram is important in computer-assisted artery motion analysis or reconstruction of 3D vascular structures from a single-plan or biplane angiographic system. Developing fully automated and accurate vessel segmentation algorithms is highly challenging, especially when extracting vascular structures with large variations in image intensities and noise, as well as with variable cross-sections or vascular lesions. Methods: This paper presents a novel tracking method for automatic segmentation of the coronary artery tree in X-ray angiographic images, based on probabilistic vessel tracking and fuzzy structure pattern inferring. The method is composed of two main steps: preprocessing and tracking. In preprocessing, multiscale Gabor filtering and Hessian matrix analysis were used to enhance and extract vessel features from the original angiographic image, leading to a vessel feature map as well as a vessel direction map. In tracking, a seed point was first automatically detected by analyzing the vessel feature map. Subsequently, two operators [e.g., a probabilistic tracking operator (PTO) and a vessel structure pattern detector (SPD)] worked together based on the detected seed point to extract vessel segments or branches one at a time. The local structure pattern was inferred by a multi-feature based fuzzy inferring function employed in the SPD. The identified structure pattern, such as crossing or bifurcation, was used to control the tracking process, for example, to keep tracking the current segment or start tracking a new one, depending on the detected pattern. Results: By appropriate integration of these advanced preprocessing and tracking steps, our tracking algorithm is able to extract both vessel axis lines and edge points, as well as measure the arterial diameters in various complicated cases. For example, it can walk across gaps along the longitudinal vessel direction, manage varying vessel curvatures, and adapt to varying vessel widths in situations with arterial stenoses and aneurysms. Conclusions: Our algorithm performs well in terms of robustness, automation, adaptability, and applicability. In particular, the successful development of two novel operators, namely, PTO and SPD, ensures the performance of our algorithm in vessel tracking.

Exploring laccase-like multicopper oxidase genes from the ascomycete Trichoderma reesei: a functional, phylogenetic and evolutionary study (2010-08-24T00:00:00Z)

Background: The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection. Results: After homologous overproduction of a candidate LMCO gene, extracellular laccase activity was detected when ABTS or SRG were used as substrates, and the recombinant protein was purified to homogeneity followed by biochemical characterization. The recombinant protein, called TrLAC1, has a molecular mass of 104 kDa. Optimal temperature and pH were respectively 40-45 degrees Celsius and 4, by using ABTS as substrate. TrLAC1 showed broad pH stability range of 3 to 7. Temperature stability revealed that TrLAC1 is not a thermostable enzyme, which was also confirmed by unfolding studies monitored by circular dichroism. Evolutionary studies were performed to shed light on the LMCO family, and the phylogenetic tree was reconstructed using maximum-likelihood method. LMCO and classical laccases were clearly divided into two distinct groups. Finally, Darwinian selection was tested, and the results showed that positive selection drove the evolution of sequences leading to well-known laccases involved in ligninolysis. Positively-selected sites were observed that could be used as targets for mutagenesis and functional studies between classical laccases and LMCO from T. reesei. Conclusions: Homologous production and evolutionary studies of the first LMCO from the biomass-degrading fungus T. reesei gives new insights into the physicochemical parameters and biodiversity in this family.

Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism (2010-08-24T00:00:00Z)

Background: Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results: Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions: 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and -oxidation of fatty acids. 3) Intracellular chemical development in A. parasiticus is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae (2010-08-20T00:00:00Z)

Background: Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from the free thiol groups of small compounds, with the reduction of a molecule of oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results: In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57degreesC, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Conclusions: Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, were found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Na+-stimulated ATPase of alkaliphilic halotolerant cyanobacterium Aphanothece halophytica translocates Na+ into proteoliposomes via Na+ uniport mechanism. (2010-08-07T00:00:00Z)

Background: When cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na+ from cells to maintain the cytoplasmic Na+ concentration. Until now, ATPase involved in Na+ transport in cyanobacteria has not been reported. Here, the characterization of ATPase and its role in Na+ transport of an alkaliphilic halotolerant Aphanothece halophytica were investigated to understand the survival mechanism of A. halophytica under high salinity condition. Results: The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na+ but not K+, Li+ and Ca2+. The apparent Km values for Na+ and ATP were 2.0 and 1.2 mM, respectively. Proteoliposomes reconstituted with the purified enzyme could take up Na+ upon the addition of ATP. The apparent Km values for this uptake were 3.3 and 0.5 mM for Na+ and ATP respectively. The mechanism of Na+ transport mediated by Na+-stimulated ATPase in A. halophytica was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na+-stimulated ATPase was observed upon the addition of ATP with Na+ but not with K+, Li+ and Ca2+. The Na+- and ATP-dependent alkalization of proteoliposome lumen was stimulated by carbonyl cyanide m - chlorophenylhydrazone (CCCP) but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na+ uptake activity. The uptake of Na+ was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na+-stimulated ATPase of the proteoliposomes. Conclusions: The data demonstrate that Na+-stimulated ATPase from A. halophytica functions as an electrogenic Na+ pump which transports only Na+ upon hydrolysis of ATP. A secondary event, Na+- and ATP-dependent H+ efflux from proteoliposomes, is driven by the electric potential generated by Na+-stimulated ATPase.

Selective control of amino acid metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae (2010-08-04T00:00:00Z)

Background: When eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. Activated Gcn2p up-regulates the general amino acid control pathway through phosphorylation of the translational initiation factor eIF2. In Saccharomyces cerevisiae, Gcn2p is the only kinase that phosphorylates eIF2 to regulate translation through this mechanism. We addressed changes in yeast growth and tRNA aminoacylation, or charging, during amino acid depletion in the presence and absence of GCN2. tRNA charging was measured using a microarray technique which simultaneously measures all cytosolic tRNAs. A fully prototrophic strain, and its isogenic gcn2 counterpart, were used to study depletion for each of the 20 amino acids, with a focus on Trp, Arg, His and Leu, which are metabolically distinct and together provide a good overview on amino acid metabolism. Results: While the wild-type strain had no observable phenotype upon depletion for any amino acid, the gcn2 strain showed slow growth in media devoid of only Trp or Arg. Consistent with the growth phenotypes, profiles of genome-wide tRNA charging revealed significant decrease in cognate tRNA charging only in the gcn2 strain upon depletion for Trp or Arg. In contrast, there was no change in tRNA charging during His and Leu depletion in either the wild-type or gcn2 strains, consistent with the null effect on growth during loss of these amino acids. We determined that the growth phenotype of Trp depletion is derived from feedback inhibition of aromatic amino acid biosynthesis. By removing Phe and Tyr from the media in addition to Trp, regular growth was restored and tRNATrp charging no longer decreased. The growth phenotype of Arg depletion is derived from unbalanced nitrogen metabolism. By supplementing ornithine upon Arg depletion, both growth and tRNAArg charging were partially restored. Conclusion: Under mild stress conditions the basal activity of Gcn2p is sufficient to allow for proper adaptation to amino acid depletion. This study highlights the importance of the GCN2 eIF2 kinase pathway for maintaining metabolic homeostasis, contributing to appropriate tRNA charging and growth adaptation in response to culture conditions deficient for the central amino acids, trypotphan and arginine.

Sequencing bias: comparison of different protocols of MicroRNA library construction (2010-09-06T00:00:00Z)

Background: MicroRNAs(miRNAs) are 18-25 nt small RNAs playing critical roles in many biological processes. The majority of known miRNAs were discovered by conventional cloning and a Sanger sequencing approach. The next-generation sequencing (NGS) technologies enable in-depth characterization of the global repertoire of miRNAs, and different protocols for miRNA library construction have been developed. However, the possible bias between the relative expression levels and sequences introduced by different protocols of library preparation have rarely been explored. Results: We assessed three different miRNA library preparation protocols, SOLiD, Illumina versions 1 and 1.5, using cloning or SBS sequencing of total RNA samples extracted from skeletal muscles from Hu sheep and Dorper sheep, and then validated 9 miRNAs by qRT-PCR. Our results show that SBS sequencing data highly correlate with Illumina cloning data. The SOLiD data, when compared to Illumina's, indicate more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- and 5'-ends, higher frequency occurrence for reads containing end secondary structure (ESS), and higher frequency for reads that do not map to known miRNAs. qRT-PCR results showed the best correlation with SOLiD cloning data. Fold difference of Hu sheep and Dorper sheep between qRT-PCR result and SBS sequencing data correlated well (r=0.937), and fold difference of miR-1 and miR-206 among SOLiD cloning data, qRT-PCR and SBS sequencing data was similar. Conclusions: The sequencing depth can influence the quantitative measurement of miRNA abundance, but the discrepancy caused by it was not statistically significant as high correlation was observed between Illumina cloning and SBS sequencing data. Bias of length distribution, sequence variation, and ESS was observed between data obtained with the different protocols. SOLiD cloning data differ from Illumina cloning data mainly because of distinct methods of adapter ligation. The good correlation between qRT-PCR result and SOLiD data might be due to the similarities of the hybridization-based methods. The fold difference analysis indicated that methods based on hybridization may be superior for quantitative measurement of miRNA abundance. Because of the genome sequence of the sheep is not available, our data may not explain how the entire miRNA bias in the natural miRNAs in sheep or other mammal miRNA expression, unbiased artificially synthesized miRNA will help on evaluating the methodology of miRNA library preparation.

Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) using filamentous fungi (2010-08-26T00:00:00Z)

Background: The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-threo-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion. Results: Keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) accumulated in the culture supernatant when Trichoderma reesei delta lga1 and Aspergillus niger delta gaaC were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate /l was produced from 20 g D-galacturonate /l and A. niger delta gaaC produced 15.0 g keto-deoxy-L-galactonate /l from 20 g polygalacturonate /l, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate per g D-galacturonate consumed. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g/l intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in A. niger delta gaaC, but decreased in T. reesei. Conclusions: Bioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both A. niger delta gaaC and T. reesei delta lga1, although production (titre, volumetric and specific rates) was better with A. niger than T. reesei. A. niger was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto-deoxy-L-galactonate accumulated intracellularly, concentrations above ~12 g/l were exported to the culture supernatant. Lysis may have contributed to the release of keto-deoxy-L-galactonate from T. reesei mycelia.

Characterization of single chain antibody targets through yeast two hybrid (2010-08-22T00:00:00Z)

Background: Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results: We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions: Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.

Oligonucleotide indexing of DNA barcodes: identification of tuna and other scombrid species in food products  (2010-08-23T00:00:00Z)

Background: DNA barcodes are a global standard for species identification and have countless applications in the medical, forensic and alimentary fields, but few barcoding methods work efficiently in samples in which DNA is degraded, e.g. foods and archival specimens. This limits the choice of target regions harbouring a sufficient number of diagnostic polymorphisms. The method described here uses existing PCR and sequencing methodologies to detect mitochondrial DNA polymorphisms in complex matrices such as foods. The reported application allowed the discrimination among 17 fish species of the Scombridae family with high commercial interest such as mackerels, bonitos and tunas which are often present in processed seafood. The approach can be easily upgraded with the release of new genetic diversity information to increase the range of detected species. Results: Cocktail of primers are designed for PCR using publicly available sequences of the target sequence. They are composed of a fixed 5' region and of variable 3' cocktail portions that allow amplification of any member of a group of species of interest. The population of short amplicons is directly sequenced and indexed using primers containing a longer 5' region and the non polymorphic portion of the cocktail portion. A 226 bp region of CytB was selected as target after collection and screening of 148 online sequences; 85 SNPs were found, of which 75 were present in at least two sequences. Primers were also designed for two shorter sub-fragments that could be amplified from highly degraded samples. The test was used on 203 samples of seafood (canned tuna and scomber, tuna salad, tuna sauce) and could successfully detect the presence of different or additional species that were not identified on the labelling of canned tuna, tuna salad and sauce samples. Conclusions: The described method is largely independent of the degree of degradation of the DNA source and can thus be applied to processed seafood. Moreover, the method is highly flexible: publicly available sequence information on mitochondrial genomes are rapidly increasing for most species, facilitating the choice of target sequences and the improvement of resolution of the test. This is particularly important for the discrimination of marine and aquaculture species for which genome information is still limited.

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation (2010-08-24T00:00:00Z)

Background: Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. Results: The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin beta-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, the capacity for glycosylation of recombinant glycoproteins expressed at very high levels, such as the magnICON-expressed E. coli enterotoxin B subunit:H.sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform. Conclusion: Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.

Does the use of the 2009 FIGO classification of endometrial cancer impact on indications of the sentinel node biopsy ?  (2010-08-30T00:00:00Z)

Background: Lymphadenectomy is debated in early stages endometrial cancer. Moreover, a new FIGO classification of endometrial cancer, merging stages IA and IB has been recently published. Therefore, the aims of the present study was to evaluate the relevance of the sentinel node (SN) procedure in women with endometrial cancer and to discuss whether the use of the 2009 FIGO classification could modify the indications for SN procedure. Methods: Eighty-five patients with endometrial cancer underwent the SN procedure followed by pelvic lymphadenectomy. SNs were detected with a dual or single labelling method in 74 and 11 cases, respectively. All SNs were analysed by both H&E staining and immunohistochemistry. Presumed stage before surgery was assessed for all patients based on MR imaging features using the 1988 FIGO classification and the 2009 FIGO classification. Results: An SN was detected in 88.2% of cases (75/85 women). Among the fourteen patients with lymph node metastases one-half were detected by serial sectioning and immunohistochemical analysis. There were no false negative case. Using the 1988 FIGO classification and the 2009 FIGO classification, the correlation between preoperative MRI staging and final histology was moderate with Kappa = 0.24 and Kappa=0.45, respectively. None of the patients with grade 1 endometrioid carcinoma on biopsy and IA 2009 FIGO stage on MR imaging exhibited positive SN. Patients with grade 2-3 endometrioid carcinoma and stage IA on MR imaging, the rate of positive SN reached 16.6% with an incidence of micrometastases of 50%. Conclusions: The present study has demonstrated that sentinel node biopsy is an adequate technique to evaluate lymph node status. The use of the 2009 FIGO classification increases the accuracy of MR imaging to stage patients with early stages of endometrial cancer and contributes to clarify the indication of SN biopsy according to tumour grade and histological type.

Bisphosphonate treatment of aggressive primary, recurrent and metastatic Giant Cell Tumour of Bone (2010-08-29T00:00:00Z)

Background: Giant cell tumour of bone (GCTB) is an expansile osteolytic tumour which contains numerous osteoclast-like giant cells. GCTB frequently recurs and can produce metastatic lesions in the lungs. Bisphosphonates are anti-resorptive drugs which act mainly on osteoclasts. Method: In this study, we have examined clinical and radiological outcomes of treatment with aminobisphosphonates on 25 cases of aggressive primary, recurrent and metastatic GCTB derived from four European centres. We also analysed in vitro the inhibitory effect of zoledronic acid on osteoclasts isolated from GCTBs. Results: Treatment protocols differed with several different aminobisphosphonates being employed, but stabilisation of disease was achieved in most of these cases which were refractory to conventional treatment. Most inoperable sacral/pelvic tumours did not increase in size and no further recurrence was seen in GCTBs that had repeatedly recurred in bone and soft tissues. Lung metastases did not increase in size or number following treatment. Zoledronic acid markedly inhibited lacunar resorption by GCTB-derived osteoclasts in vitro. Conclusion: Our findings suggest that bisphosphonates may be useful in controlling disease progression in GCTB and that these agents directly inhibit GCTB - derived osteoclast resorption. These studies highlight the need for the establishment of standardised protocols to assess the efficacy of bisphosphonate treatment of GCTB.

CYP1A1 gene polymorphisms increase lung cancer risk in a high-incidence region of Spain: a case control study (2010-08-30T00:00:00Z)

Background: A rural region in south-west Spain has one of the highest lung cancer incidence rates of the country, as revealed by a previous epidemiological 10-year follow-up study. The present work was undertaken to ascertain the role of CYP1A1 gene polymorphisms and their interaction with tobacco smoking in the development of the disease in this location. Methods: One-hundred-and-three cases of lung cancer and 265 controls participated in the study. The participants were screened for the presence of four CYP1A1 polymorphisms, namely MspI, Ile462Val, T3205C, and Thr461Asn. Lung cancer risk was estimated as odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression models adjusting for age, sex, and smoking. Results: The distribution of the variant CYP1A1 alleles was different from that described for other Caucasian populations, with CYP1A1*2A showing an uncommonly high frequency (p< 0.01). The CYP1A1*2B allele (carrying MspI and Ile462Val mutations) was strongly associated with high lung cancer risk (OR=4.59, CI:1.4-12.6, p<0.01). The Ile462Val polymorphism was also shown to increase the risk for the disease (OR=4.51, CI:1.8-11.9; p<0.01) and particularly for squamous-cell (OR=5.01; CI: 1.6-14.3, p<0.01) and small-cell lung carcinoma (SCLC) (OR = 6.97, CI: 1.2-81.3; p=0.04). Moreover, the Thr461Asn polymorphism was found to be associated with SCLC in a Caucasian population for the first time to our knowledge (OR=8.33, CI: 1.3-15.2, p=0.04). Conclusion: The results suggest that CYP1A1 polymorphisms contribute to increase lung cancer susceptibility in an area with an uncommon high incidence rate.

COX-2 inhibition improves immunotherapy and is associated with decreased numbers of myeloid-derived suppressor cells in mesothelioma  (2010-08-30T00:00:00Z)

Background: : Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that accumulates in tumour-bearing hosts. These cells are induced by tumour-derived factors (e.g. prostaglandins) and have a critical role in immune suppression. MDSC suppress T and NK cell function via increased expression of arginase I and production of reactive oxygen species (ROS) and nitric oxide (NO). Immune suppression by MDSC was found to be one of the main factors for immunotherapy insufficiency. Here we investigate if the in vivo immunoregulatory function of MDSC can be reversed by inhibiting prostaglandin synthesis by specific COX-2 inhibition focussing on ROS production by MDSC subtypes. In addition, we determined if dietary celecoxib treatment leads to refinement of immunotherapeutic strategies.MethodS: MDSC numbers and function were analysed during tumour progression in a murine model for mesothelioma. Mice were inoculated with mesothelioma tumour cells and treated with cyclooxygenase-2 (COX-2) inhibitor celecoxib, either as single agent or in combination with dendritic cell-based immunotherapy. Results: : We found that large numbers of infiltrating MDSC co-localise with COX-2 expression in those areas where tumour growth takes place. Celecoxib reduced prostaglandin E2 levels in vitro and in vivo. Treatment of tumour-bearing mice with dietary celecoxib prevented the local and systemic expansion of all MDSC subtypes. The function of MDSC was impaired as was noticed by reduced levels of ROS and NO and reversal of T cell tolerance; resulting in refinement of immunotherapy. Conclusions: : We conclude that celecoxib is a powerful tool to improve dendritic cell-based immunotherapy and is associated with a reduction in the numbers and suppressive function of MDSC. These data suggest that immunotherapy approaches benefit from simultaneously blocking cyclooxygenase-2 activity.

Significance of Aurora B overexpression in hepatocellular carcinoma (2010-08-28T00:00:00Z)

Background: To investigate the significance of Aurora B expression in hepatocellular carcinoma (HCC). Methods: The Aurora B and Aurora A mRNA level was measured in 160 HCCs and the paired nontumorous liver tissues by reverse transcription-polymerase chain reaction. Mutations of the p53 and beta-catenin genes were analyzed in 134 and 150 tumors, respectively, by direct sequencing of exon 2 to exon 11 of p53 and exon 3 of beta-catenin. Anticancer effects of AZD1152-HQPA, an Aurora B kinase selective inhibitor, were examined in Huh-7 and Hep3B cell lines. Results: Aurora B was overexpressed in 98 (61%) of 160 HCCs and in all 7 HCC cell lines examined. The overexpression of Aurora B was associated with Aurora A overexpression (P = 0.0003) and p53 mutation (P = 0.002) and was inversely associated with beta-catenin mutation (P = 0.002). Aurora B overexpression correlated with worse clinicopathologic characteristics. Multivariate analysis confirmed that Aurora B overexpression was an independent poor prognostic factor, despite its interaction with Aurora A overexpression and mutations of p53 and beta-catenin. In Huh-7 and Hep3B cells, AZD1152-HQPA induced proliferation blockade, histone H3 (Ser10) dephosphorylation, cell cycle disturbance, and apoptosis. Conclusion: Aurora B overexpression is an independent molecular marker predicting tumor invasiveness and poor prognosis of HCC. Aurora B kinase selective inhibitors are potential therapeutic agents for HCC treatment.

Hypoxia stimulates the expression of macrophage migration inhibitory factor in human vascular smooth muscle cells via HIF-1alpha dependent pathway (2010-08-20T00:00:00Z)

Background: Hypoxia plays an important role in vascular remodeling and directly affects vascular smooth muscle cells (VSMC) functions. Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor, and recent evidence suggests an important role of MIF in the progression of atherosclerosis and restenosis. However, the potential link between hypoxia and MIF in VSMC has not been investigated. The current study was designed to test whether hypoxia could regulate MIF expression in human VSMC. The effect of modulating MIF expression on hypoxia-induced VSMC proliferation and migration was also investigated at the same time. Results: Expression of MIF mRNA and protein was up-regulated as early as 2 hours in cultured human VSMCs after exposed to moderate hypoxia condition (3% O2). The up-regulation of MIF expression appears to be dependent on hypoxia-inducible transcription factor-1 alpha (HIF-1 alpha) since knockdown of HIF-1alpha inhibits the hypoxia induction of MIF gene and protein expression. The hypoxia induced expression of MIF was attenuated by antioxidant treatment as well as by inhibition of extracellular signal-regulated kinase (ERK). Under moderate hypoxia conditions (3% O2), both cell proliferation and cell migration were increased in VSMC cells. Blocking the MIF by specific small interference RNA to MIF (MIF-shRNA) resulted in the suppression of proliferation and migration of VSMCs. Conclusion: Our results demonstrated that in VSMCs, hypoxia increased MIF gene expression and protein production. The hypoxia-induced HIF-1 alpha activation, reactive oxygen species (ROS) generation and ERK activation might be involved in this response. Both MIF and HIF-1alpha mediated the hypoxia response of vascular smooth muscle cells, including cell migration and proliferation.

Karyopherin alpha7 (KPNA7), a divergent member of the importin alpha family of nuclear import receptors (2010-08-11T00:00:00Z)

Background: Classical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin alpha and importin beta. NLS cargo is recognized by importin alpha, which is bound by importin beta. Importin beta mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin beta triggers disassembly of the complex. To date, six importin alpha family members, encoded by separate genes, have been described in humans. Results: We sequenced and characterized a seventh member of the importin alpha family of transport factors, karyopherin alpha 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin beta (IBB) is divergent, and shows stronger binding to importin beta than the IBB domains from of other importin alpha family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic. Conclusion: KPNA7 is a novel importin alpha family member in humans that belongs to the importin alpha 2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin alpha family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.

AlphaS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form (2010-08-12T00:00:00Z)

Background: Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. Results: In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both alphaS1- and beta-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature beta-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature alphaS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of alphaS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of alphaS1-casein with membranes. Conclusions: These experiments reveal for the first time the existence of a membrane-associated form of alphaS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that alphaS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway.

HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation (2010-08-11T00:00:00Z)

Background: High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results: We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as Myod, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions: Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.

Lamellipodium extension and membrane ruffling require different SNARE-mediated trafficking pathways  (2010-08-10T00:00:00Z)

Background: Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling. Results: We report the first description of a SNARE complex, containing SNAP23, syntaxin13 and cellubrevin/VAMP3, that is induced by cell adhesion to an extracellular matrix. Impairing the function of the SNAREs in the complex using inhibitory SNARE domains disrupted the recycling endosome, impeded delivery of integrins to the cell surface, and reduced haptotactic cell migration and spreading. Blocking SNAP23 also inhibited the formation of PMA-stimulated, F-actin-rich membrane ruffles; however, membrane ruffle formation was not significantly altered by inhibition of VAMP3 or syntaxin13. In contrast, membrane ruffling, and not cell spreading, was sensitive to inhibition of two SNAREs within the biosynthetic secretory pathway, GS15 and VAMP4. Consistent with this, formation of a complex containing VAMP4 and SNAP23 was enhanced by treatment of cells with PMA. The results reveal a requirement for the function of a SNAP23-syntaxin13-VAMP3 complex in the formation of lamellipodia during cell adhesion and of a VAMP4-SNAP23-containing complex during PMA-induced membrane ruffling. Conclusions: Our findings suggest that different SNARE-mediated trafficking pathways support membrane remodeling during ECM-induced lamellipodium extension and PMA-induced ruffle formation, pointing to important mechanistic differences between these processes.

Use of the GenoType(R) MTBDRplus assay to assess drug resistance of Mycobacterium tuberculosis isolates from patients in rural Uganda  (2010-08-06T00:00:00Z)

Background: Drug resistance levels and patterns among Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients in Mbarara Uganda were investigated. Methods: We enrolled, consecutively, all newly diagnosed and previously treated smear-positive TB patients aged [greater than or equal to] 18 years. Isolates were tested for drug resistance against rifampicin (RIF) and isoniazid (INH) using the Genotype(R) MDRTBplus assay and results were compared with those obtained by the indirect proportion method on Lowenstein-Jensen media. HIV testing was performed using two rapid HIV tests. Results: A total of 125 isolates from 167 TB suspects with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analysed. A majority (92.8%) of the participants were newly presenting while only 7.2% were retreatment cases. Resistance mutations to either RIF or INH were detected in 6.4% of the total isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpobeta gene mutations seen in the sample were D516V, S531L, H526Y H526D and D516V, while one strain had a Delta1 mutation in the wild type probes. There were three strains with katG (codon 315) gene mutations while only one strain showed the inhA promoter region gene mutation. Conclusion: The TB resistance rate in Mbarara is relatively low. The GenoType(R) MTBDRplus assay can be used for rapid screening of MDR-TB in this setting.

Accumulation of advanced glycation end (AGEs) products in intensive care patients: an observational, prospective study  (2010-05-25T00:00:00Z)

Background: Oxidative stress plays an important role in the course and eventual outcome in a majority of patients admitted to the intensive care unit (ICU). Markers to estimate oxidative stress are not readily available in a clinical setting. AGEs accumulation has been merely described in chronic conditions, but can also occur acutely due to oxidative stress. Since AGEs have emerged to be stable end products, these can be a marker of oxidative stress. Skin autofluorescence (AF) is a validated marker of tissue content of AGEs. We hypothesized that AGEs accumulate acutely in ICU patients. Methods: We performed an observational prospective study in a medical surgical ICU in a university affiliated teaching hospital. All consecutively admitted ICU patients in a 2 month period were included. Skin AF was measured using an AGE reader in 35 consecutive ICU patients > 18 yrs. As a comparison, historical data of a control group (n = 231) were used. These were also used to calculate age-adjusted AF-levels (AFadj). Values are expressed as median and interquartile range [P25-P75]. Differences between groups were tested by non parametric tests. P<0.05 was considered statistically significant. Results: AFadj values were higher in ICU patients (0.33 [0.00 - 0.68]) than in controls (-0.07 [-0.29 - 0.24]; P<0.001). No differences in skin AFadj were observed between acute or planned admissions, or presence of sepsis, nor was skin AFadj related to severity of disease as estimated by APACHE-II score, length of ICU, hospital stay or mortality. Conclusion: Acute AGE accumulation in ICU patients was shown in this study, although group size was small. This can possibly reflect oxidative stress in ICU patients. Further studies should reveal whether AGE-accumulation will be a useful parameter in ICU patients and whether skin AF has a predictive value for outcome, which was not shown in this small study.

Comparison of haematological parameters determined by the Sysmex KX - 2IN automated haematology analyzer and the manual counts (2010-04-23T00:00:00Z)

Background: This study was designed to determine the correlation between heamatological parameters by Sysmex KX-21N automated hematology analyzer with the manual methods.MethodSixty (60) subjects were randomly selected from both apparently healthy subjects and those who have different blood disorders from the University of Teaching Hospital (UNTH), Ituku-Ozalla, Enugu, Enugu State, Nigeria. Three (3)mls of venous blood sample was collected aseptically from each subject into tri-potassium ethylenediamine tetra-acetic acid (K3EDTA) for the analysis of haematological parameters using the automated and the manual methods. Results: The blood film report by the manual method showed that 50% of the subjects were normocytic-normochromic while the other 50% revealed different abnormal blood pictures. Also, there were statistically significant differences (p<0.05) in mean cell hemoglobin concentrations (MCHC) between the two methods. Similarly, the mean (S.E) values of hemoglobin, packed cell volume, platelet and total white cell counts demonstrated statistically significant difference (p<0.001) and correlated positively when both methods were compared. Conclusion: From the present study, it can be concluded that the automated hematology analyzer readings correlated well with readings by the standard manual method, although the latter method gave additional diagnostic information on the blood pictures. While patients' care and laboratory operations could be optimized by using manual microscopic examination as a reflective substitute for automated methods, usage of automated method would ease our workload and save time for patients.

The combined positive impact of Lean methodology and Ventana Symphony autostainer on histology lab workflow (2010-02-08T00:00:00Z)

Background: Histologic samples all funnel through the H&E microtomy staining area. Here manual processes intersect with semi-automated processes creating a bottleneck. We compare alternate work processes in anatomic pathology primarily in the H&E staining work cell. Methods: We established a baseline measure of H&E process impact on personnel, information management and sample flow from historical workload and production data and direct observation. We compared this to performance after implementing initial LEAN process modifications, including workstation reorganization, equipment relocation and workflow levelling, and the Ventana Symphony stainer to assess the impact on productivity in the H&E staining work cell. Results: Average time from gross station to assembled case decreased by 2.9 hours (12%). Total process turnaround time (TAT) exclusive of processor schedule changes decreased 48 minutes/case (4%). Mean quarterly productivity increased 8.5% with the new methods. Process redesign reduced the number of manual steps from 219 to 182, a 17% reduction. Specimen travel distance was reduced from 773 ft/case to 395 ft/case (49%) overall, and from 92 to 53 ft/case in the H&E cell (42% improvement). Conclusions: Implementation of Lean methods in the H&E work cell of histology can result in improved productivity, improved through-put and case availability parameters including TAT.

SNAI1 expression and the mesenchymal phenotype: an immunohistochemical study performed on 46 cases of oral squamous cell carcinoma  (2010-02-05T00:00:00Z)

Background: SNAI1 can initiate epithelial-mesenchymal transition (EMT), leading to loss of epithelial characteristics and, in cancer, to invasion and metastasis. We hypothesized that SNAI1 reactivation occurs in oral squamous cell carcinoma (OSCC) where it might also be associated with focal adhesion kinase (FAK) expression and p63 loss. Methods: Immunohistochemistry was performed on 46 tumors and 26 corresponding lymph node metastases. Full tissue sections were examined to account for rare and focal expression. Clinical outcome data were collected and analyzed. Results: SNAI1-positivity (nuclear, greater than or equal to 5% tumor cells) was observed in 10 tumors and 5 metastases (n=12 patients). Individual SNAI1(+) tumor cells were seen in primary tumors of 30 patients. High level SNAI1 expression (>10% tumor cells) was rare, but significantly associated with poor outcome. Two cases displayed a sarcomatoid component as part of the primary tumor with SNAI1(+)/FAK(+)/E-cadherin(-)/p63(-) phenotype, but disparate phenotypes in corresponding metastases. All cases had variable SNAI1(+) stroma. A mesenchymal-like immunoprofile in primary tumors characterized by E-cadherin loss (n=29, 63%) or high cytoplasmic FAK expression (n=10, 22%) was associated with N(+) status and tumor recurrence/new primary, respectively. Conclusions: SNAI1 is expressed, although at low levels, in a substantial proportion of OSCC. High levels of SNAI1 may herald a poor prognosis and circumscribed SNAI1 expression can indicate the presence of a sarcomatoid component. Absence of p63 in this context does not exclude squamous tumor origin. Additional EMT inducers may contribute to a mesenchymal-like phenotype and OSCC progression.

Mapping Haplotype-haplotype Interactions with Adaptive LASSO (2010-08-27T00:00:00Z)

Background: The genetic etiology of complex diseases in human has been commonly viewed as a complex process involving both genetic and environmental factors functioning in a complicated manner. Quite often the interactions among genetic variants play major roles in determining the susceptibility of an individual to a particular disease. Statistical methods for modeling interactions underlying complex diseases between single genetic variants (e.g. single nucleotide polymorphisms or SNPs) have been extensively studied. Recently, haplotype-based analysis has gained its popularity among genetic association studies. When multiple sequence or haplotype interactions are involved in determining an individual's susceptibility to a disease, it presents daunting challenges in statistical modeling and testing of the interaction effects, largely due to the complicated higher order epistatic complexity. Results: In this article, we propose a new strategy in modeling haplotype-haplotype interactions under the penalized logistic regression framework with adaptive L1-penalty. We consider interactions of sequence variants between haplotype blocks. The adaptive L1-penalty allows simultaneous effect estimation and variable selection in a single model. We propose a new parameter estimation method which estimates and selects parameters by the modified Gauss-Seidel method nested within the EM algorithm. Simulation studies show that it has low false positive rate and reasonable power in detecting haplotype interactions. The method is applied to test haplotype interactions involved in mother and offspring genome in a small for gestational age (SGA) neonates data set, and significant interactions between different genomes are detected. Conclusions: As demonstrated by the simulation studies and real data analysis, the approach developed provides an efficient tool for the modeling and testing of haplotype interactions. The implementation of the method in R codes can be freely downloaded from http://www.stt.msu.edu/~cui/software.html.

A haplotype inference algorithm for trios based on deterministic sampling (2010-08-23T00:00:00Z)

Background: In genome-wide association studies, thousands of individuals are genotyped in hundreds of thousands of single nucleotide polymorphisms (SNPs). Statistical power can be increased when haplotypes, rather than three-valued genotypes, are used in analysis, so the problem of haplotype phase inference (phasing) is particularly relevant. Several phasing algorithms have been developed for data from unrelated individuals, based on different models, some of which have been extended to father-mother-child "trio" data. Results: We introduce a technique for phasing trio datasets using a tree-based deterministic sampling scheme. We have compared our method with publicly available algorithms PHASE v2.1, BEAGLE v3.0.2 and 2SNP v1.7 on datasets of varying number of markers and trios. We have found that the computational complexity of PHASE makes it prohibitive for routine use; on the other hand 2SNP, though the fastest method for small datasets, was significantly inaccurate. We have shown that our method outperforms BEAGLE in terms of speed and accuracy for small to intermediate dataset sizes in terms of number of trios for all marker sizes examined. Our method is implemented in the "Tree-Based Deterministic Sampling" (TDS) package, available for download at www.ee.columbia.edu/~anastas/tds Conclusions: Using a Tree-Based Deterministic sampling technique, we present an intuitive and conceptually simple phasing algorithm for trio data. The trade off between speed and accuracy achieved by our algorithm makes it a strong candidate for routine use on trio datasets.

Population structure and genetic bottleneck in sweet cherry estimated with SSRs and the gametophytic self-incompatibility locus (2010-08-20T00:00:00Z)

Background: Domestication and breeding involve the selection of particular phenotypes, limiting the genetic diversity of the population and creating a bottleneck. These effects can be precisely estimated when the location of domestication is established. Few analyses have focused on understanding the genetic consequences of domestication and breeding in fruit trees. In this study, we aimed to analyse genetic structure and changes in the diversity in sweet cherry Prunus avium L. Results: Three subgroups were detected in sweet cherry, with one group of landraces genetically very close to the analysed wild cherry population. A limited number of SSR markers displayed deviations from the frequencies expected under neutrality. After the removal of these markers from the analysis, a very limited bottleneck was detected between wild cherries and sweet cherry landraces, with a much more pronounced bottleneck between sweet cherry landraces and modern sweet cherry varieties. The loss of diversity between wild cherries and sweet cherry landraces at the S-locus was more significant than that for microsatellites. Particularly high levels of differentiation were observed for some S-alleles. Conclusions: Several domestication events may have happened in sweet cherry or/and intense gene flow from local wild cherry was probably maintained along the evolutionary history of the species. A marked bottleneck due to breeding was detected, with all markers, in the modern sweet cherry gene pool. The microsatellites did not detect the bottleneck due to domestication in the analysed sample. The vegetative propagation specific to some fruit trees may account for the differences in diversity observed at the S-locus. Our study provides insights into domestication events of cherry, however, requires confirmation on a larger sampling scheme for both sweet cherry landraces and wild cherry.

Co-assortment in integron-associated gene cassette assemblages in environmental DNA samples. (2010-08-10T00:00:00Z)

Background: It has been shown that integron-associated gene cassettes exist largely in tandem arrays of variable size, ranging from antibiotic resistance arrays of three to five cassettes up arrays of more than 100 cassettes associated with the vibrios. Further, the ecology of the integron / gene cassette system has been investigated by showing that very many different cassettes are present in even small environmental samples. In this study, we seek to extend the ecological perspective on the integron / gene cassette system by investigating the way in which this diverse cassette metagenome is apportioned amongst prokaryote lineages in a natural environment. Results: We used a combination of PCR based techniques, applied to environmental DNA samples, with ecological analytical techniques to establish co-assortment within cassette populations and then established the relationship between this co-assortment and genomic structures. We then assessed the distribution of gene cassettes within the environment and found that the majority of gene cassettes existed in large co-assorting groups. Conclusions: Our results suggested that the gene cassette diversity of a relatively pristine sampling environment was structured into co-assorting groups, predominantly containing large numbers of cassettes per group. These co-assorting groups consisted of different gene cassettes in stoichiometric relationship. Conservatively, we then attributed co-assorting cassettes to the gene cassette complements of single prokaryote lineages and by implication, to large integron associated arrays. The prevalence of large arrays in the environment raises new questions about the assembly, maintenance and utility of large cassette arrays in prokaryote populations.

Microsatellite-based genetic diversity and population structure of domestic sheep in northern Eurasia (2010-08-10T00:00:00Z)

Background: Identification of global livestock diversity hotspots and their importance in diversity maintenance is essential for making global conservation efforts. We screened 52 sheep breeds from the Eurasian subcontinent with 20 microsatellite markers. By estimating and weighting differently within- and between-breed genetic variation our aims were to identify genetic diversity hotspots and prioritize the importance of each breed for conservation, respectively. In addition we estimated how important within-species diversity hotspots are in livestock conservation. Results: Bayesian clustering analysis revealed three genetic clusters, termed Nordic, Composite and Fat-tailed. Southern breeds from close to the region of sheep domestication were more variable, but less genetically differentiated compared with more northern populations. Decreasing weight for within-breed diversity component led to very high representation of genetic clusters or regions containing more diverged breeds, but did not increase phenotypic diversity among the high ranked breeds. Sampling populations throughout 14 regional groups was suggested for maximized total genetic diversity. Conclusions: During initial steps of establishing a livestock conservation program populations from the diversity hot-spot area are the most important ones, but for the full design our results suggested that approximately equal population presentation across environments should be considered. Even in this case, higher per population emphasis in areas of high diversity is appropriate. The analysis was based on neutral data, but we have no reason to think the general trend is limited to this type of data. However, a comprehensive valuation of populations should balance production systems, phenotypic traits and available genetic information, and include consideration of probability of success.

Comparison of information-theoretic to statistical methods for gene-environment interactions in the presence of genetic heterogeneity (2010-09-03T00:00:00Z)

Background: Multifactorial diseases such as cancer and cardiovascular diseases are caused by the complex interplay between genes and environment. The detection of these interactions remains challenging due to computational limitations. Information theoretic approaches use computationally efficient directed search strategies and thus provide a feasible solution to this problem. However, the power of information theoretic methods for interaction analysis has not been systematically evaluated. In this work, we compare power and Type I error of an information-theoretic approach to existing interaction analysis methods. Methods: The k-way interaction information (KWII) metric for identifying variable combinations involved in gene-gene interactions (GGI) was assessed using several simulated data sets under models of genetic heterogeneity driven by susceptibility increasing loci with varying allele frequency, penetrance values and heritability. The power and proportion of false positives of the KWII was compared to multifactor dimensionality reduction (MDR), restricted partitioning method (RPM) and logistic regression. Results: The power of the KWII was considerably greater than MDR on all six simulation models examined. For a given disease prevalence at high values of heritability, the power of both RPM and KWII was greater than 95%. For models with low heritability and/or genetic heterogeneity, the power of the KWII was consistently greater than RPM; the improvements in power for the KWII over RPM ranged from 4.7% to 14.2% at for alpha = 0.001 in the three models at the lowest heritability values examined. KWII performed similar to logistic regression. Conclusions: Information theoretic models are flexible and have excellent power to detect GGI under a variety of conditions that characterize complex diseases.

Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes (2010-09-02T00:00:00Z)

Background: Discontinuous genes have been observed in bacteria, archaea, and eukaryotic nuclei, mitochondria and chloroplasts. Gene discontinuity occurs in multiple forms: the two most frequent result from introns that are spliced out of the RNA and the resulting exons are spliced together to form a single transcript, and fragmented gene transcripts that are not covalently attached post-transcriptionally. Within the past few years, fragmented ribosomal RNA (rRNA) genes have been discovered in bilateral metazoan mitochondria, all within a group of related oysters. Results: In this study, we have characterized this fragmentation with comparative analysis and experimentation. We present secondary structures, modeled using comparative sequence analysis of the discontinuous mitochondrial large subunit rRNA genes of the cupped oysters C. virginica, C. gigas, and C. hongkongensis. Comparative structure models for the large subunit rRNA in each of the three oyster species are generally similar to those for other bilateral metazoans. We also used RT-PCR and analyzed ESTs to determine if the two fragmented LSU rRNAs are spliced together. The two segments are transcribed separately, and not spliced together although they still form functional rRNAs and ribosomes. Conclusions: Although many examples of discontinuous ribosomal genes have been documented in bacteria and archaea, as well as the nuclei, chloroplasts, and mitochondria of eukaryotes, oysters are some of the first characterized examples of fragmented bilateral animal mitochondrial rRNA genes. The secondary structures of the oyster LSU rRNA fragments have been predicted on the basis of previous comparative metazoan mitochondrial LSU rRNA structure models.

Expression of immune-response genes in lepidopteran host are suppressed by venom from an endoparasitoid, Pteromalus puparum (2010-09-02T00:00:00Z)

Background: The relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids are widely used as biological control agents in sustainable agricultural programs. Females of the gregarious endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae) inject venom along with eggs into their hosts. P. puparum does not inject polydnaviruses during oviposition. For this reason, P. puparum and its pupal host, the small white butterfly Pieris rapae (Lepidoptera: Pieridae), comprise an excellent model system for studying the influence of an endoparasitoid venom on the biology of the pupal host. P. puparum venom suppresses the immunity of its host, although the suppressive mechanisms are not fully understood. In this study, we tested our hypothesis that P. puparum venom influences host gene expression in the two main immunity-conferring tissues, hemocytes and fat body. Results: At 1 h post-venom injection, we recorded significant decreases in transcript levels of 217 EST clones (revealing 113 genes identified in silico, including 62 unknown contigs) derived from forward subtractive libraries of host hemocytes and in transcript levels of 288 EST clones (221 genes identified in silico, including 123 unknown contigs) from libraries of host fat body. These genes are related to insect immune response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, stress response and transcriptional and translational regulation. We verified the reliability of the suppression subtractive hybridization (SSH) data with semi-quantitative RT-PCR analysis of a set of randomly selected genes. This analysis showed that most of the selected genes were down-regulated after venom injection. Conclusions: Our findings support our hypothesis that P. puparum venom influences gene expression in host hemocytes and fat body. Specifically, the venom treatments led to reductions in expression of a large number of genes. Many of the down-regulated genes act in immunity, although others act in non-immune areas of host biology. We conclude that the actions of venom on host gene expression influence immunity as well as other aspects of host biology in ways that benefit the development and emergence of the next generation of parasitoids.

Evolution of duplicated IgH loci in Atlantic salmon, Salmo salar (2010-09-02T00:00:00Z)

Background: The Atlantic salmon (Salmo salar) immunoglobulin heavy chain (IgH) locus possesses two parallel IgH isoloci (IGH-A and IGH-B), that are related to the genomic duplication event in the family Salmonidae. These duplicated IgH loci in Atlantic salmon provide a unique opportunity to examine the mechanisms of genome diversity and genome evolution of the IgH loci in vertebrates. In this study, we defined the structure of these loci in Atlantic salmon, and sequenced 24 bacterial artificial chromosome (BAC) clones that were assembled into the IGH-A (1.1Mb) and IGH-B (0.9 Mb) loci. In addition, over 7,000 cDNA clones from the IgH variable (VH) region have been sequenced and analyzed. Results: The present study shows that the genomic organization of the duplicated IgH loci in Atlantic salmon differs from that in other teleosts and other vertebrates. The loci possess multiple Ctau genes upstream of the Cmu region, with three of the Ctau genes being functional. Moreover, the duplicated loci possess over 300 VH segments which could be classified into 18 families. This is the largest number of VH families currently defined in any vertebrate. There were significant structural differences between the two loci, indicating that both IGH-A and -B loci have evolved independently in the short time after the recent genome duplication approximately 60 mya. Conclusions: Our results indicate that the duplication of the IgH loci in Atlantic salmon significantly contributes to the increased diversity of the antibody repertoire, as compared with the single IgH locus in other vertebrates.

DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster (Crassostrea gigas) (2010-08-27T00:00:00Z)

Background: DNA methylation is an epigenetic mechanism with important regulatory functions in animals. While the mechanism itself is evolutionarily ancient, the distribution and function of DNA methylation is diverse both within and among phylogenetic groups. Although DNA methylation has been well studied in mammals, there are limited data on invertebrates, particularly molluscs. Here we characterize the distribution and investigate potential functions of DNA methylation in the Pacific oyster (Crassostrea gigas). Results: Methylation sensitive PCR and bisulfite sequencing PCR approaches were used to identify CpG methylation in C.gigas genes and demonstrated that this species possesses intragenic methylation. In silico analysis of CpGo/e ratios in publicly available sequence data suggests that DNA methylation is a common feature of the C.gigas genome, and that specific functional categories of genes have significantly different levels of methylation. Conclusions: The Pacific oyster genome displays intragenic DNA methylation and contains genes necessary for DNA methylation in animals. Results of this investigation suggest that DNA methylation has regulatory functions in Crassostrea gigas, particularly in gene families that have inducible expression, including those involved in stress and environmental responses.

Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases  (2010-08-20T00:00:00Z)

Background: Complement C2 deficiency is the most common genetically determined complete complement deficiency and is associated with a number of diseases. Most prominent are the associations with recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults. The links with these diseases reflect the important role complement C2 plays in both innate immunity and immune tolerance. Infusions with normal fresh frozen plasma for the treatment of associated disease have demonstrated therapeutic effects but so far protein replacement therapy has not been evaluated. Results: Human complement C2 was cloned and expressed in a mammalian cell line. The purity of recombinant human C2 (rhC2) was greater than 95% and it was characterized for stability and activity. It was sensitive to C1s cleavage and restored classical complement pathway activity in C2-deficient serum both in a complement activation ELISA and a hemolytic assay. Furthermore, rhC2 could increase C3 fragment deposition on the human pathogen Streptococcus pneumoniae in C2-deficient serum to levels equal to those with normal serum. Conclusions: Taken together these data suggest that recombinant human C2 can restore classical complement pathway activity and may serve as a potential therapeutic for recurring bacterial infections or SLE in C2-deficient patients.

Ultrastructural localization of extracellular matrix proteins of the lymph node cortex: evidence supporting the reticular network as a pathway for lymphocyte migration  (2010-08-17T00:00:00Z)

Background: The lymph node (LN) is a crossroads of blood and lymphatic vessels allowing circulating lymphocytes to efficiently recognize foreign molecules displayed on antigen presenting cells. Increasing evidence indicates that after crossing high endothelial venules, lymphocytes migrate within the node along the reticular network (RN), a scaffold of fibers enwrapped by fibroblastic reticular cells (FRC). Light microscopy has shown that the RN contains specific extracellular matrix (ECM) proteins, which are putative molecular "footholds" for migration, and are known ligands for lymphocyte integrin adhesion receptors. Results: To investigate whether ECM proteins of the RN are present on the outer surface of the FRC and are thus accessible to migrating lymphocytes, ultrastructural immunohistochemical staining of cynomolgus monkey LN was performed using antibodies to human ECM proteins that were successfully employed at the light microscopic level. The fibrillar collagens I and III were observed primarily within the reticular network fibers themselves. In contrast, the matrix proteins laminin, fibronectin, collagen IV, and tenascin were observed within the reticular fibers and also on the outer membrane surface of the FRC. Conclusions: These findings suggest a molecular basis for how the RN functions as a pathway for lymphocyte migration within the lymph node.

Dynamics of dendritic cell maturation are identified through a novel filtering strategy applied to biological time-course microarray replicates (2010-08-03T00:00:00Z)

Background: Dendritic cells (DC) play a central role in primary immune responses and become potent stimulators of the adaptive immune response after undergoing the critical process of maturation. Understanding the dynamics of DC maturation would provide key insights into this important process. Time course microarray experiments can provide unique insights into DC maturation dynamics. Replicate experiments are necessary to address the issues of experimental and biological variability. Statistical methods and averaging are often used to identify significant signals. Here a novel strategy for filtering of replicate time course microarray data, which identifies consistent signals between the replicates, is presented and applied to a DC time course microarray experiment. Results: The temporal dynamics of DC maturation were studied by stimulating DC with poly(I:C) and following gene expression at 5 time points from 1 to 24 hours. The novel filtering strategy uses standard statistical and fold change techniques, along with the consistency of replicate temporal profiles, to identify those differentially expressed genes that were consistent in two biological replicate experiments. To address the issue of cluster reproducibility a consensus clustering method, which identifies clusters of genes whose expression varies consistently between replicates, was also developed and applied. Analysis of the resulting clusters revealed many known and novel characteristics of DC maturation, such as the up-regulation of specific immune response pathways. Intriguingly, more genes were down-regulated than up-regulated. Results identify a more comprehensive program of down-regulation, including many genes involved in protein synthesis, metabolism, and housekeeping needed for maintenance of cellular integrity and metabolism. Conclusions: The new filtering strategy emphasizes the importance of consistent and reproducible results when analyzing microarray data and utilizes consistency between replicate experiments as a criterion in both feature selection and clustering, without averaging or otherwise combining replicate data. Observation of a significant down-regulation program during DC maturation indicates that DC are preparing for cell death and provides a path to better understand the process. This new filtering strategy can be adapted for use in analyzing other large-scale time course data sets with replicates.

Peripheral blood T Regulatory cell counts may not predict transplant rejection (2010-07-15T00:00:00Z)

Background: Recent evidence shows that allograft survival rates show a positive correlation with the number of circulating T regulatory cells (Tregs). This study investigated both the number and the cytokine profiles exhibited by Foxp3+ Tregs in blood, spleen and lymph nodes of Lewis rat recipients of BN rat cardiac allografts after a single-dose of Rapamycin (RAPA). Results: Rats were divided into three groups: control group ( containing healthy control and acute rejection group), and recipients treated with a single dose of RAPA on either Day 1 (1D group)or Day 3 (3D group) post-transplant. We analyzed the number of Foxp3+Tregs and the expression of Foxp3 and cytokines in the peripheral blood and the peripheral lymphoid tissues. No difference was found in the numbers of circulating Foxp3+ Tregs between these three groups. RAPA administration significantly increased Foxp3 expression in peripheral lymphoid tissues after a single dose of RAPA on Day 3 post-transplant. Foxp3+Tregs inhibited the activity of effector T cells (Teff) via the secretion of TGF-beta1. Conclusion: The number of Tregs in the recipient's blood may not be a good predictor of transplant rejection. Foxp3+Tregs inhibit the activity of Teff cells mainly in the peripheral lymphoid tissues.

Changes in colon gene expression associated with increased colon inflammation in interleukin-10 gene-deficient mice inoculated with Enterococcus species (2010-07-15T00:00:00Z)

Background: Inappropriate responses to normal intestinal bacteria may be involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohn's Disease (CD), Ulcerative Colitis (UC)) and variations in the host genome may mediate this process. IL-10 gene-deficient (Il10-/-) mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains, and have therefore been used as an animal model of CD. Comprehensive characterization of changes in cecum gene expression levels associated with inflammation in the Il10-/- mouse model has recently been reported. Our aim was to characterize changes in colonic gene expression levels in Il10-/- and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EF.CIF). We investigated two hypotheses: (1) that oral inoculation of Il10-/- mice would result in greater and more consistent intestinal inflammation than that observed in Il10-/- mice not receiving this inoculation, and (2) that this inflammation would be associated with changes in colon gene expression levels similar to those previously observed in human studies, and these mice would therefore be an appropriate model for human CD. Results: At 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44k mouse arrays. Differentially expressed genes were identified using linear models for microarray analysis (Bioconductor), and these genes were clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in Il10-/- mice as a result of inoculation, with the strongest effect being in the EF and EF.CIF groups. Genes differentially expressed in Il10-/- mice as a result of EF or EF.CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes). Conclusions: Our results suggest that colonic inflammation in Il10-/- mice inoculated with solutions containing Enterococcus strains is associated with gene expression changes similar to those of human IBD, specifically CD, and that with the EF.CIF inoculum in particular this is an appropriate model to investigate food-gene interactions relevant to human CD.

Use of fuzzy edge single-photon emission computed tomography analysis in definite Alzheimer's disease - a retrospective study (2010-09-01T00:00:00Z)

Background: Definite Alzheimer's disease (AD) requires neuropathological confirmation. Single-photon emission computed tomography (SPECT) may enhance diagnostic accuracy, but due to restricted sensitivity and specificity, the role of SPECT is largely limited with regard to this purpose. Methods: We propose a new method of SPECT data analysis. The method is based on a combination of parietal lobe selection (as regions-of-interest (ROI)), 3D fuzzy edge detection, and 3D watershed transformation. We applied the algorithm to three-dimensional SPECT images of human brains and compared the number of watershed regions inside the ROI between AD patients and controls. The Student's two-sample t-test was used for testing domain number equity in both groups. Results: AD patients had a significantly reduced number of watershed regions compared to controls (p < 0.01). A sensitivity of 94.1% and specificity of 80% was obtained with a threshold value of 57.11 for the watershed domain number. The narrowing of the SPECT analysis to parietal regions leads to a substantial increase in both sensitivity and specificity. Conclusions: Our non-invasive, relatively low-cost, and easy method can contribute to a more precise diagnosis of AD.

Rapid T1 Quantification based on 3D Phase Sensitive Inversion Recovery  (2010-08-17T00:00:00Z)

Background: In Contrast Enhanced Magnetic Resonance Imaging fibrotic myocardium can be distinguished from healthy tissue using the difference in the longitudinal T1 relaxation after administration of Gadolinium, the so-called Late Gd Enhancement. The purpose of this work was to measure the myocardial absolute T1 post-Gd from a single breath-hold 3D Phase Sensitivity Inversion Recovery sequence (PSIR). Equations were derived to take the acquisition and saturation effects on the magnetization into account. Methods: The accuracy of the method was investigated on phantoms and using simulations. The method was applied to a group of patients with suspected myocardial infarction where the absolute difference in relaxation of healthy and fibrotic myocardium was measured at about 15 minutes post-contrast. The evolution of the absolute R1 relaxation rate (1 / T1) over time after contrast injection was followed for one patient and compared to T1 mapping using Look-Locker. Based on the T1 maps synthetic LGE images were reconstructed and compared to the conventional LGE images. Results: The fitting algorithm is robust against variation in acquisition flip angle, the inversion delay time and cardiac arrhythmia. The observed relaxation rate of the myocardium is 1.2 s-1, increasing to 6 - 7 s-1 after contrast injection and decreasing to 2 - 2.5 s-1 for healthy myocardium and to 3.5 - 4 s-1 for fibrotic myocardium. Synthesized images based on the T1 maps correspond very well to actual LGE images. Conclusions: The method provides a robust quantification of post-Gd T1 relaxation for a complete cardiac volume within a single breath-hold.

Evaluation of both perfusion and atrophy in multiple system atrophy of the cerebellar type using brain SPECT alone (2010-08-11T00:00:00Z)

Background: Partial volume effects in atrophied areas should be taken into account when interpreting brain perfusion single photon emission computed tomography (SPECT) images of neurodegenerative diseases. To evaluate both perfusion and atrophy using brain SPECT alone, we developed a new technique applying tensor-based morphometry (TBM) to SPECT. Methods: After linear spatial normalization of brain perfusion SPECT using 99mTc-ethyl cysteinate dimer (99mTc-ECD) to a Talairach space, high-dimension-warping was done using an original 99mTc-ECD template. Contraction map images calculated from Jacobian determinants and spatially normalized SPECT images using this high-dimension-warping were compared using statistical parametric mapping (SPM2) between two groups of 16 multiple system atrophy of the cerebellar type (MSA-C) patients and 73 age-matched normal controls. This comparison was also performed in conventionally warped SPECT images. Results: SPM2 demonstrated statistically significant contraction indicating local atrophy and decreased perfusion in the whole cerebellum and pons of MSA-C patients as compared to normal controls. Higher significance for decreased perfusion in these areas was obtained in high-dimension-warping than in conventional warping, possibly due to sufficient spatial normalization to a 99mTc-ECD template in high-dimensional warping of severely atrophied cerebellum and pons. In the present high-dimension-warping, modification of tracer activity remained within 3% of the original tracer distribution. Conclusions: The present new technique applying TBM to brain SPECT provides information on both perfusion and atrophy at the same time thereby enhancing the role of brain perfusion SPECT in clinical studies of neurodegenerative diseases.

Reliability of computed tomography measurements in assessment of thigh muscle cross-sectional area and attenuation  (2010-08-11T00:00:00Z)

Background: Advancement in technology of computer tomography (CT) and introduction of new medical imaging softwares enables easy and rapid assessment of muscle cross-sectional area (CSA) and attenuation. Before using these techniques in clinical studies there is a need for evaluation of the reliability of the measurements. The purpose of the study was to evaluate the inter- and intra-observer reliability of ImageJ in measuring thigh muscles CSA and attenuation in patients with anterior cruciate ligament (ACL) injury by computer tomography. Methods: 31 patients from an ongoing study of rehabilitation and muscle atrophy after ACL reconstruction were included in the study. Axial CT images with slice thickness of 10 mm at the level of 150 mm above the knee joint were analyzed by two investigators independently at two times with a minimum of 3 weeks between the two readings using NIH ImageJ. CSA and the mean attenuation of individual thigh muscles were analyzed for both legs. Results: Mean CSA and mean attenuation values were in good agreement both when comparing the two observers and the two replicates. The inter- and intraclass correlation (ICC) was generally very high with values from 0.98 to 1.00 for all comparisons except for the area of semimembranosus. All the ICC values were significant (p<0,001). Pearson correlation coefficients were also generally very high with values from 0.98 to 1.00 for all comparisons except for the area of semimembranosus (0.95 for intraobserver and 0.92 for interobserver). Conclusion: This study has presented ImageJ as a method to monitor and evaluate CSA and attenuation of different muscles in the thigh using CT-imaging. The method shows an overall excellent reliability with respect to both observer and replicate.

An open-source software tool for the generation of relaxation time maps in magnetic resonance imaging (2010-07-30T00:00:00Z)

Background: In magnetic resonance (MR) imaging, T1, T2 and T2* relaxation times represent characteristic tissue properties that can be quantified with the help of specific imaging strategies. While there are basic software tools for specific pulse sequences, until now there is no universal software program available to automate pixel-wise mapping of relaxation times from various types of images or MR systems. Such a software program would allow researchers to test and compare new imaging strategies and thus would significantly facilitate research in the area of quantitative tissue characterization. Results: After defining requirements for a universal MR mapping tool, a software program named MRmap was created using a high-level graphics language. Additional features include a manual registration tool for source images with motion artifacts and a tabular DICOM viewer to examine pulse sequence parameters. MRmap was successfully tested on three different computer platforms with image data from three different MR system manufacturers and five different sorts of pulse sequences: multi-image inversion recovery T1; Look-Locker/ TOMROP T1; modified Look-Locker inversion recovery (MOLLI) T1; single-echo T2/ T2*; and multi-echo T2/ T2*. Computing times varied between 2 and 113 seconds. Estimates of relaxation times compared favorably to those obtained from non-automated curve fitting. Completed maps were exported in DICOM format and could be read in standard software packages used for analysis of clinical and research MR data. Conclusions: MRmap is a flexible cross-platform research tool that enables accurate mapping of relaxation times from various pulse sequences. The software allows researchers to optimize quantitative MR strategies in a manufacturer-independent fashion. The program and its source code were made available as open-source software on the internet.

Multiple imputation for estimation of an occurrence rate in cohorts with attrition and discrete follow-up time points: a simulation study (2010-09-03T00:00:00Z)

Background: In longitudinal cohort studies, subjects may be lost to follow-up at any time during the study. This leads to attrition and thus to a risk of inaccurate and biased estimations. The purpose of this paper is to show how multiple imputation can take advantage of all the information collected during follow-up in order to estimate the cumulative probability P(E) of an event E, when the first occurrence of this event is observed at t successive time points of a longitudinal study with attrition. Methods: We compared the performance of multiple imputation with that of Kaplan-Meier estimation in several simulated attrition scenarios. Results: In missing-completely-at-random scenarios, the multiple imputation and Kaplan-Meier methods performed well in terms of bias (less than 1%) and coverage rate (range=[94.4%; 95.8%]). In missing-at-random scenarios, the Kaplan-Meier method was associated with a bias ranging from -5.1% to 7.0% and with a very poor coverage rate (as low as 0.2%). Multiple imputation performed much better in this situation (bias <2%, coverage rate >83.4%). Conclusions: Multiple imputation shows promise for estimation of an occurrence rate in cohorts with attrition. This study is a first step towards defining appropriate use of multiple imputation in longitudinal studies.

Sampling and coverage issues of telephone surveys used for collecting health information in Australia: results from a face-to-face survey from 1999 to 2008 (2010-08-26T00:00:00Z)

Background: To examine the trend of "mobile only" households, and households that have a mobile phone or landline telephone listed in the telephone directory, and to describe these groups by various socio-demographic and health indicators.MethodRepresentative face-to-face population health surveys of South Australians, aged 15 years and over, were conducted in 1999, 2004, 2006, 2007 and 2008 (n=14285, response rates=51.9% to 70.6%). Self-reported information on mobile phone ownership and usage (1999 to 2008) and listings in White Pages telephone directory (2006 to 2008), and landline telephone connection and listings in the White Pages (1999 to 2008), was provided by participants. Additional information was collected on self-reported health conditions and health-related risk behaviours. Results: Mobile only households have been steadily increasing from 1.4% in 1999 to 8.7% in 2008. In terms of sampling frame for telephone surveys, 68.7% of South Australian households in 2008 had at least a mobile phone or landline telephone listed in the White Pages (73.8% in 2006; 71.5% in 2007). The proportion of mobile only households was highest among young people, unemployed, people who were separated, divorced or never married, low income households, low SES areas, rural areas, current smokers, current asthma or people in the normal weight range. The proportion with landlines or mobiles telephone numbers listed in the White Pages telephone directory was highest among older people, married or in a defacto relationship or widowed, low SES areas, rural areas, people classified as overweight, or those diagnosed with arthritis or osteoporosis. Conclusion: The rate of mobile only households has been increasing in Australia and is following worldwide trends, but has not reached the high levels seen internationally (12% to 52%). In general, the impact of mobile telephones on current sampling frames (exclusion or non-listing of mobile only households or not listed in the White Pages directory) may have a low impact on health estimates obtained using telephone surveys. However, researchers need to be aware that mobile only households are distinctly different to households with a landline connection, and the increase in the number of mobile-only households is not uniform across all groups in the community. Listing in the White Pages directory continues to decrease and only a small proportion of mobile only households are listed. Researchers need to be aware of these telephone sampling issues when considering telephone surveys.

Interpretation of evidence in data by untrained medical students: a scenario-based study (2010-08-26T00:00:00Z)

Background: To determine which approach to assessment of evidence in data - statistical tests or likelihood ratios - comes closest to the interpretation of evidence by untrained medical students. Methods: Empirical study of medical students (N=842), untrained in statistical inference or in the interpretation of diagnostic tests. They were asked to interpret a hypothetical diagnostic test, presented in four versions that differed in the distributions of test scores in diseased and non-diseased populations. Each student received only one version. The intuitive application of the statistical test approach would lead to rejecting the null hypothesis of no disease in version A, and to accepting the null in version B. Application of the likelihood ratio approach led to opposite conclusions - against the disease in A, and in favour of disease in B. Version C tested the importance of the p-value (A: 0.04 versus C: 0.08) and version D the importance of the likelihood ratio (C: 1/4 versus D: 1/8). Results: In version A, 7.5% concluded that the result was in favour of disease (compatible with p value), 43.6% ruled against the disease (compatible with likelihood ratio), and 48.9% were undecided. In version B, 69.0% were in favour of disease (compatible with likelihood ratio), 4.5% against (compatible with p value), and 26.5% undecided. Increasing the p value from 0.04 to 0.08 did not change the results. The change in the likelihood ratio from 1/4 to 1/8 increased the proportion of non-committed responses. Conclusions: Most untrained medical students appear to interpret evidence from data in a manner that is compatible with the use of likelihood ratios.

Identifying nurse staffing research in Medline: development and testing of empirically derived search strategies with the PubMed interface (2010-08-23T00:00:00Z)

Background: The identification of health services research in databases such as PubMed/Medline is a cumbersome task. This task becomes even more difficult if the field of interest involves the use of diverse methods and data sources, as is the case with nurse staffing research. This type of research investigates the association between nurse staffing parameters and nursing and patient outcomes. A comprehensively developed search strategy may help identify nurse staffing research in PubMed/Medline. Methods: A set of relevant references in PubMed/Medline was identified by means of three systematic reviews. This development set was used to detect candidate free-text and MeSH terms. The frequency of these terms was compared to a random sample from PubMed/Medline in order to identify terms specific to nurse staffing research, which were then used to develop a sensitive, precise and balanced search strategy. To determine their precision, the newly developed search strategies were tested against a) the pool of relevant references extracted from the systematic reviews, b) a reference set identified from an electronic journal screening, and c) a sample from PubMed/Medline. Finally, all newly developed strategies were compared to PubMed's Health Services Research Queries (PubMed's HSR Queries). Results: The sensitivities of the newly developed search strategies were almost 100% in all of the three test sets applied; precision ranged from 6.1% to 32.0%. PubMed's HSR queries were less sensitive (83.3% to 88.2%) than the new search strategies. Only minor differences in precision were found (5.0% to 32.0%). Conclusions: As with other literature on health services research, nurse staffing studies are difficult to identify in PubMed/Medline. Depending on the purpose of the search, researchers can choose between high sensitivity and retrieval of a large number of references or high precision, i.e. and an increased risk of missing relevant references, respectively. More standardized terminology (e.g. by consistent use of the term "nurse staffing") could improve the precision of future searches in this field. Empirically selected search terms can help to develop effective search strategies. The high consistency between all test sets confirmed the validity of our approach.

Determining the interviewer effect on CQ Index outcomes: a multilevel approach  (2010-08-19T00:00:00Z)

Background: The CQ Index for the elderly, a quality-of-care questionnaire administered by conducting interviews, is used to assess clients' experiences in Dutch nursing homes and homes for the elderly. This article describes whether inter-interviewer differences influence the perceived quality of healthcare services reported by residents, the size of this interviewer effect and the influence of the interviewer characteristics on CQ Index dimensions for public reporting. Methods: Data from 4345 questionnaires was used. Correlations were calculated, reliability analyses were performed, and a multilevel analysis was used to calculate the degree of correlation between two interviewers within one health care institution. Five models were constructed and the Intra Class Correlation (ICC) was calculated. Healthcare institutions were given 1-5 stars on every quality dimensions (1 = worst and 5 = best), adjusted for resident and interviewer characteristics. The effect of these characteristics on the assignment of the stars was investigated. Results: In a multilevel approach, the ICC showed a significant amount of variance on five quality dimensions. Of the interviewer characteristics, only previous interviewing experience, the reason of interviewing and general knowledge of health care had a significant effect on the quality dimensions. Adjusting for interviewer characteristics did not affect the overall star assignment to the institutions regarding 7 of 12 quality dimensions. For the other five dimensions (Shared decision-making, Meals, Professional competency, Autonomy, and Availability of personnel) a minor effect was found. Conclusions: We have shown that training, the use of experienced interviewers, written instructions, supervision and educational meetings do not automatically prevent interviewer effects. While the results of this study can be used to improve the quality of services provided by these institutions, several CQ index dimensions should be interpreted with caution for external purposes (accountability and transparency).

Two storage hexamerins from the beet armyworm Spodoptera exigua: Cloning, characterization and the effect of gene silencing on survival (2010-08-31T00:00:00Z)

Background: In insects, hemocyanin superfamily proteins accumulate apparently to serve as sources of amino acids during metamorphosis, reproduction and development. Storage hexamerins are important members of the hemocyanin superfamily. Although insects possess storage hexamerins, very little is known about the character and specific functions of hexamerin 1 and storage protein 1 in insect development. Results: To gain insight into the function of storage proteins in insects, cDNAs for two storage proteins were cloned from the fat body of Spodoptera exigua. S. exigua hexamerin 1 (SeHex) cDNA contained an open reading frame of 2124 nucleotides encoding a protein of 707 amino acids with a predicted molecular weight of 82.12 kDa. S. exigua storage protein 1 (SeSP1) cDNA contained an open reading frame of 2256 bp encoding a protein of 751 amino acids with a predicted molecular weight of ~88.84 kDa. Northern blotting analyses revealed that SeHex mRNA is expressed in the fat body, cuticle, midgut and Malpighian tubules and SeSP1 in fat body, Malpighian tubules and tracheae. SeHex and SeSP1 mRNAs were expressed in fat body at different levels from first instar larvae to pupae, with expression was much lower from first instar larvae to first-day fifth instar larvae. SeHex transcript expression was high in fat body of wandering larvae (pre-pupae) and steadily decreased to the seventh pupal day. SeSP1 transcript expression was high in fat body of wandering larvae, 2-day-old fifth instar larvae and 2-, 4- and 7-day-old pupae. SeHex and SeSP1 mRNAs levels were expressed lower than control on the condition of starvation at 12 h . Of insects injected with SeHex and SeSP1 dsRNA, 38.7% and 24.3% survived to 204 h after treatment, respectively. This was significantly lower than in the controls groups. Conclusions: These findings provide new data on the tissue distribution, expression patterns and the function in starvation of storage proteins. RNA interference results revealed that storage protein genes are key in metamorphosis, reproduction and insect development. The results for SeHex and SeSP1 interference reveal that a potential method to control this pest is to disrupt the regulation of storage proteins.

EGF regulates survivin stability through the Raf-1/ERK pathway in insulin-secreting pancreatic beta-cells  (2010-08-31T00:00:00Z)

Background: Postnatal expansion of the pancreatic beta-cell mass is required to maintain glucose homeostasis immediately after birth. This beta-cell expansion is regulated by multiple growth factors, including glucose, insulin, insulin-like growth factor (IGF-1) and epidermal growth factor (EGF). These mitogens signal through several downstream pathways (AKT, ERK, STAT3, and JNK) to regulate the survival and proliferation of betacells. Survivin, an oncofetal protein with both pro-proliferative and anti-apoptotic properties, is a known transcriptional target of both IGF-1 and EGF in cancer cells. Here, we analyzed the effects of the beta-cell mitogens IGF-1 and EGF on survivin regulation in the established pancreatic beta-cell model cell lines, MIN6 and INS-1 and in primary mouse islets. Results: In pancreatic beta-cells, treatment with glucose, insulin, or EGF increased survivin protein levels at early time points. By contrast, no significant effects on survivin were observed following IGF-1 treatment. EGF-stimulated increases in survivin protein were abrogated in the presence of downstream inhibitors of the Raf-1/MEK/ERK pathway. EGF had no significant effect on survivin transcription however it prolonged the half-life of the survivin protein and stabilized survivin protein levels by inhibiting survivin ubiquitination. Conclusions: This study defines a novel mechanism of survivin regulation by EGF through the Raf-1/MEK/ERK pathway in pancreatic beta-cells, via prolongation of survivin protein half-life and inhibition of the ubiquitin-mediated proteasomal degradation pathway. This mechanism may be important for regulating beta-cell expansion after birth.

The chicken miR-150 targets the avian orthologue of the functional zebrafish MYB 3'UTR target site (2010-09-02T00:00:00Z)

Background: The c-myb proto-oncogene is the founding member of a family of transcription factors involved principally in haematopoiesis, in diverse organisms, from zebrafish to mammals. Its deregulation has been implicated in human leukaemogenesis and other cancers. The expression of c-myb is tightly regulated by post-transcriptional mechanisms involving microRNAs. MicroRNAs are small, highly conserved non-coding RNAs that inhibit translation and decrease mRNA stability by binding to regulatory motifs mostly located in the 3'UTR of target mRNAs conserved throughout evolution. MYB is an evolutionarily conserved miR-150 target experimentally validated in mice, humans and zebrafish. However, the functional miR-150 sites of humans and mice are orthologous, whereas that of zebrafish is different. Results: We identified the avian mature miRNA-150-5P, Gallus gallus gga-miR-150 from chicken leukocyte small-RNA libraries and showed that, as expected, the gga-miR-150 sequence was highly conserved, including the seed region sequence present in the other miR-150 sequences listed in miRBase. Reporter assays showed that gga-miR-150 acted on the avian MYB 3'UTR and identified the avian MYB target site involved in gga-miR-150 binding. A comparative in silico analysis of the miR-150 target sites of MYB 3'UTRs from different species led to the identification of a single set of putative target sites in amphibians and zebrafish, whereas two sets of putative target sites were identified in chicken and mammals. However, only the target site present in the chicken MYB 3'UTR that was identical to that in zebrafish was functional, despite the additional presence of mammalian target sites in chicken. This specific miR-150 site usage was not cell-type specific and persisted when the chicken c-myb 3'UTR was used in the cell system to identify mammalian target sites, showing that this miR-150 target site usage was intrinsic to the chicken c-myb 3'UTR. Conclusion: Our study of the avian MYB/gga-miR-150 interaction shows a conservation of miR-150 target site functionality between chicken and zebrafish that does not extend to mammals.

L1CAM expression in endometrial carcinomas is regulated by usage of two different promoter regions (2010-08-27T00:00:00Z)

Background: The L1 cell adhesion molecule (L1CAM) was originally identified as a neural adhesion molecule involved in axon guidance. In many human epithelial carcinomas L1CAM is overexpressed and thereby augments cell motility, invasion and metastasis formation. L1CAM positive carcinomas are associated with bad prognosis. Recent data point out that L1CAM is regulated in a fashion similar to epithelial-mesenchymal transition (EMT). Previous studies have implied the transcription factors Slug and/or beta-catenin in L1CAM transcriptional regulation. However, the regulation of human L1CAM expression at the transcriptional level is not well understood. Results: To better understand the molecular basis of L1CAM transcriptional regulation, we carried out a detailed characterization of the human L1CAM promoter. We identified two transcription start sites, the first in front of a non-translated exon 0 (promoter 1) and the other next to the first protein-coding exon 1 (promoter 2). Both sites could be verified in endometrial carcinoma (EC) cell lines and appear to be used in a cell-type specific manner. The two identified promoter regions showed activity in luciferase reporter assays. Chromatin-IP analyses confirmed the in silico predicted E-boxes, binding sites for transcription factors Snail and Slug, as well as Lef-1 sites, which are related to beta-catenin-mediated transcriptional regulation, in both promoters. Overexpression of beta-catenin exclusively augmented activity of promoter 1 whereas Slug enhanced promoter 1 and 2 activity suggesting that both promoters can be active. Overexpression of beta-catenin or Slug could upregulate L1CAM expression in a cell-type specific manner. Conclusions: Our results, for the first time, provide evidence that the L1CAM gene has two functionally active promoter sites that are used in a cell-type specific manner. Slug and beta-catenin are involved L1CAM transcriptional regulation. Nevertheless, Slug rather than beta-catenin levels are correlated with L1CAM expression in EC cell lines. Our findings suggest that the L1CAM transcriptional regulation is more complex than anticipated and this study provides the basis for a better understanding of L1CAM regulation in non-neuronal/tumor cells.

Functional and cellular characterization of human Retinoic Acid Induced 1 (RAI1) mutations associated with Smith-Magenis Syndrome (2010-08-25T00:00:00Z)

Background: Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1. Results: We generated the full-length cDNA of the wild type protein and five mutated forms: RAI1-HA 2687delC, RAI1-HA 3103delC, RAI1 R960X, RAI1-HA Q1562R, and RAI1-HA S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1.Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity. Conclusion: Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains.

Quantification of vestibular-induced eye movements in zebrafish larvae (2010-09-03T00:00:00Z)

Background: Vestibular reflexes coordinate movements or sensory input with changes in body or head position. Vestibular-evoked responses that involve the extraocular muscles include the vestibulo-ocular reflex (VOR), a compensatory eye movement to stabilize retinal images. Although an angular VOR attributable to semicircular canal stimulation was reported to be absent in free-swimming zebrafish larvae, recent studies reveal that vestibular-induced eye movements can be evoked in zebrafish larvae by both static tilts and dynamic rotations that tilt the head with respect to gravity. Results: We have determined herein the basis of sensitivity of the larval eye movements with respect to vestibular stimulus, developmental stage, and sensory receptors of the inner ear. For our experiments, video recordings of larvae rotated sinusoidally at 0.25 Hz were analyzed to quantitate eye movements under infrared illumination. We observed a robust response that appeared as early as 72 hours post fertilization (hpf), which increased in amplitude over time. Unlike rotation about an earth horizontal axis, rotation about an earth vertical axis at 0.25 Hz did not evoke eye movements. Moreover, vestibular-induced responses were absent in mutant cdh23 larvae and larvae lacking anterior otoliths. Conclusions: Our results provide evidence for a functional vestibulo-oculomotor circuit in 72 hpf zebrafish larvae that relies upon sensory input from anterior/utricular otolith organs.

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation. (2010-09-01T00:00:00Z)

Background: Human beta-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the beta-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Results: Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels, Conclusions: cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

Gap junctions in olfactory neurons modulate olfactory sensitivity (2010-08-27T00:00:00Z)

Background: One of the fundamental questions in olfaction is whether olfactory receptor neurons (ORNs) behave as independent entities within the olfactory epithelium. On the basis that mature ORNs express multiple connexins, I postulated that gap junctional communication modulates olfactory responses in the periphery and that disruption of gap junctions in ORNs reduces olfactory sensitivity. The data collected from characterizing connexin 43 (Cx43) dominant negative transgenic mice OlfDNCX, and from calcium imaging of wild type mice (WT) support my hypothesis. Results: I generated OlfDNCX mice that express a dominant negative Cx43 protein, Cx43/beta-gal, in mature ORNs to inactivate gap junctions and hemichannels composed of Cx43 or other structurally related connexins. Characterization of OlfDNCX revealed that Cx43/beta-gal was exclusively expressed in areas where mature ORNs resided. Real-time quantitative PCR indicated that cellular machineries of OlfDNCX were normal in comparison to WT. Electroolfactogram recordings showed decreased olfactory responses to octaldehyde, heptaldehyde and acetyl acetate in OlfDNCX compared to WT. Octaldehyde-elicited glomerular activity in the olfactory bulb, measured according to odor-elicited c-fos mRNA upregulation in juxtaglomerular cells, was confined to smaller areas of the glomerular layer in OlfDNCX compared to WT. In WT mice, octaldehyde sensitive neurons exhibited reduced response magnitudes after application of gap junction uncoupling reagents and the effects were specific to subsets of neurons. Conclusions: My study has demonstrated that altered assembly of Cx43 or structurally related connexins in ORNs modulates olfactory responses and changes olfactory activation maps in the olfactory bulb. Furthermore, pharmacologically uncoupling of gap junctions reduces olfactory activity in subset ORNs. These data suggest that gap junctional communication or hemichannel activity plays a critical role in maintaining olfactory sensitivity and odor perception.

Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response (2010-08-26T00:00:00Z)

Background: Ciguatoxins (CTXs) are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO) and molecular pathway enrichment of the gene expression data. Results: A pronounced 4 degrees C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p<0.0001) with microarray results. Conclusions: Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against neuroinflammation. Pathologic activity of the complement/coagulation cascade has been shown in patients suffering from a chronic form of ciguatera poisoning and is of particular interest in this model. Anti-inflammatory processes were at work not only in the brain but were also seen in whole blood and liver of these animals, creating a systemic anti-inflammatory environment to protect against the initial cellular damage caused by the toxin.

Watching TV news as a memory task -- brain activation and age effects (2010-08-26T00:00:00Z)

Background: Neuroimaging studies which investigate brain activity underlying declarative memory processes typically use artificial, unimodal laboratory stimuli. In contrast, we developed a paradigm which much more closely approximates real-life situations of information encoding. Methods: In this study, we tested whether ecologically valid stimuli - clips of a TV news show - are apt to assess memory-related fMRI activation in healthy participants across a wide age range (22-70 years). We contrasted brain responses during natural stimulation (TV news video clips) with a control condition (scrambled versions of the same clips with reversed audio tracks). After scanning, free recall performance was assessed. Results: The memory task evoked robust activation of a left-lateralized network, including primarily lateral temporal cortex, frontal cortex, as well as the left hippocampus. Further analyses revealed that - when controlling for performance effects - older age was associated with greater activation of left temporal and right frontal cortex. Conclusion: We demonstrate the feasibility of assessing brain activity underlying declarative memory using a natural stimulation paradigm with high ecological validity. The preliminary result of greater brain activation with increasing age might reflect an attempt to compensate for decreasing episodic memory capacity associated with aging.

Factors associated with whole carcass condemnation rates in provincially-inspected abattoirs in Ontario 2001-2007: implications for food animal syndromic surveillance (2010-08-12T00:00:00Z)

Background: Ontario provincial abattoirs have the potential to be important sources of syndromic surveillance data for emerging diseases of concern to animal health, public health and food safety. The objectives of this study were to: (1) describe provincially inspected abattoirs processing cattle in Ontario in terms of the number of abattoirs, the number of weeks abattoirs process cattle, geographical distribution, types of whole carcass condemnations reported, and the distance animals are shipped for slaughter; and (2) identify various seasonal, secular, disease and non-disease factors that might bias the results of quantitative methods, such as cluster detection methods, used for food animal syndromic surveillance. Results: Data were collected from the Ontario Ministry of Agriculture, Food and Rural Affairs and the Ontario Cattlemen's Association regarding whole carcass condemnation rates for cattle animal classes, abattoir compliance ratings, and the monthly sales-yard price for various cattle classes from 2001-2007. To analyze the association between condemnation rates and potential explanatory variables including abattoir characteristics, season, year and commodity price, as well as animal class, negative binomial regression models were fit using generalized estimating equations (GEE) to account for autocorrelation among observations from the same abattoir. Results of the fitted model found animal class, year, season, price, and audit rating are associated with condemnation rates in Ontario abattoirs. In addition, a subset of data was used to estimate the average distance cattle are shipped to Ontario provincial abattoirs. The median distance from the farm to the abattoir was approximately 82 km, and 75% of cattle were shipped less than 100 km. Conclusions: The results suggest that secular and seasonal trends, as well as some non-disease factors will need to be corrected for when applying quantitative methods for syndromic surveillance involving these data. This study also demonstrated that animals shipped to Ontario provincial abattoirs come from relatively local farms, which is important when considering the use of spatial surveillance methods for these data.

Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic? (2010-08-04T00:00:00Z)

In a recent paper written by Hilbe et al (BMC vet res, 2009), the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE) were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we revisit these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject.Here we come back on our material as it is possible to study and demonstrate the specificity of prion immunodetection using the PET-Blot method (Paraffin Embedded Tissue - Blot). It is admitted that this method allows detecting the Proteinase K (PK) resistant form of the abnormal prion protein (PrPres) without any confusion with unspecific immunoreaction. We re-analysed the kidney tissue versus adrenal gland and brain samples from the same cheetah affected with TSE using this PET-Blot method. The PET-Blot analysis revealed specific PrPres detection within the brain, adrenal gland and some glomeruli of the kidney, with a complete identicalness compared to our previous detection using immunohistochemistry. In conclusion, these new data enable us to confirm with assurance the presence of specific abnormal prion protein in the adrenal gland and in the kidney of the cheetah affected with FSE. It also emphasizes the usefulness for the re-examination of any available tissue blocks with the PET-Blot method as a sensitive complementary tool in case of doubtful PrP IHC results.

Effect of the feeding system on the fatty acid composition, expression of the Delta9-desaturase, Peroxisome Proliferator-Activated Receptor Alpha, Gamma, and Sterol Regulatory Element Binding Protein 1 genes in the semitendinous muscle of light lambs (2010-07-22T00:00:00Z)

Background: Conjugated linoleic acids (CLAs) are receiving increasing attention because of their beneficial effects on human health, with milk and meat products derived from ruminants as important sources of CLA in the human diet. SCD gene is responsible for some of the variation in CLA concentration in adipose tissues, and PPARgamma, PPARalpha and SREBP1 genes are regulator of SCD gene. The aim of this work was to evaluate the effect of the feeding system on fatty acid composition, CLA content and relative gene expression of delta 9-desaturase (SCD), Peroxisome Proliferator-Activated Receptor Gamma (PPARgamma), Peroxisome Proliferator-Activated Receptor Alpha (PPARalpha) and Sterol Regulatory Element Binding Protein (SREBP1) in Rasa Aragonesa light lambs in semitendinous muscle. Forty-four single-born male lambs were used to evaluate the effect of the feeding system, varying on an intensity gradient according to the use of concentrates: 1. grazing alfalfa, 2. grazing alfalfa with a supplement for lambs, 3. indoor lambs with grazing ewes and 4. drylot. Results: Both grazing systems resulted in a higher concentration of vaccenic acid (VA), CLA, CLA/ VA acid ratio, and a lower oleic content, oleic acid (C18:1)/ stearic acid (C18:0) ratio, PUFA n-6/ n-3 ratio and SCD expression compared to other diets. In addition feeding system affected the fatty acid composition and SCD expression, possibly due to CLA concentration or the PUFA n-6/ n-3 ratio. Both expression of the SCD gene and the feeding system were important factors affecting CLA concentration in the animal's semitendinous muscle. PPARgamma, PPARalpha and SREBP1 expression seemed to be unaffected by the feeding system. Although no significant results were found, PPARgamma, PPARalpha and SREBP1 showed similar expression pattern as SCD. Moreover, the correlation results between SCD expression and PPARgamma (p<0.01), as well as SREBP1 (p<0.01) expression, may suggest that these genes were affecting SCD expression in a different way. Conclusions: The data indicated that the feeding system is the main factor affecting the fatty acid composition and SCD gene expression, which is also affected by CLA and possibly by n-6/ n-3 PUFAs.

Effect of abrupt weaning at housing on leukocyte distribution, functional activity of neutrophils, and acute phase protein response of beef calves (2010-07-22T00:00:00Z)

Background: Sixteen, spring-born, single suckled, castrated male calves of Limousin x Holstein-Friesian and Simmental x Holstein-Friesian dams respectively, were used to investigate the effect of weaning on total leukocyte and differential counts, neutrophil functional activity, lymphocyte immunophenotypes, and acute phase protein response. Calves grazed with their dams until the end of the grazing season when they were housed in a slatted floor shed. On the day of housing, calves were assigned to a treatment, (i) abruptly weaned (W: n = 8) or (ii) non-weaned (controls) (C: n = 8). Weaned calves were housed in pens without their dams, whereas non-weaned (control) calves were housed with their dams. Blood was collected on day -7, 0 (housing), 2, 7, and 14 to determine total leukocyte and differential counts and concentration of fibrinogen and haptoglobin. Lymphocyte immunophenotypes were characterised using selected surface antigens (CD4+, CD8+, WC1+ (gamma delta T cells), MHC Class II+ lymphocytes), and the functional activities of neutrophils (surface expression of L-selectin (CD62L), phagocytic and oxidative burst activity) were investigated using flow cytometry. Results: Treatment x sampling time interactions (P < 0.05) were detected for total leukocyte and neutrophil counts, all lymphocyte subsets, mean fluorescence intensity of CD62L+ neutrophils, and percentage neutrophils performing phagocytosis. On d 2, total leukocyte and neutrophil counts increased (P < 0.001), and percentage CD4+ and CD8+ lymphocytes, mean fluorescence intensity of CD62L+ neutrophils decreased (P < 0.05) in W compared with baseline, whereas they were unchanged (P > 0.05) in C. On d 2, percentage WC1+ lymphocytes and percentage phagocytic neutrophils decreased (P < 0.05), whereas percentage MHC class II+ lymphocytes increased (P < 0.05) in W and C, however the magnitude of change was greater in W than C. There were no treatment x sampling time interactions (P > 0.05) for monocyte, eosinophil, and basophil counts, percentage G1+ neutrophils, or percentage oxidative burst positive neutrophils. Conclusions: Abrupt weaning resulted in increased neutrophil count and impaired trafficking and phagocytic function. Together with the changes in lymphocyte subsets, the results suggest that there was a greater transitory reduction in immune function at housing in abruptly weaned than non-weaned beef calves.

Characterisation of physiological and immunological responses in beef cows to abrupt weaning and subsequent housing (2010-07-20T00:00:00Z)

Background: Weaning involves the permanent separation of the calf from the dam and has been shown to be stressful for both. The objectives of this study were to characterise the effect of i) abrupt weaning and ii) subsequent housing on the extended physiological and immunological responses of beef cows. At weaning (day (d) 0, mean age of calf (s.d.) 212 (24.5) d), cows were abruptly separated from their calves and returned to the grazing area. After 35 d at pasture, cows were housed in a slatted floor shed and offered grass silage ad libitum plus a mineral-vitamin supplement daily. Rectal body temperature was recorded and blood samples were obtained on i) d 0 (weaning), 2, 7, 14, 21, 28, 35 and subsequently on ii) d 0 (housing), 2, 7, 14 and 21 for physiological, haematological and immunological measurements. Results: Post-weaning, concentration of cortisol and dehydroepiandrosterone were unchanged (P>0.05). Rectal body temperature, neutrophil number and neutrophil: lymphocyte ratio increased (P<0.01) on d 2 compared with pre-weaning baseline. Lymphocyte and neutrophil number decreased (P<0.05) on d 2 to 7 and d 7 to 21, respectively, compared with pre-weaning baseline. Interferon-gamma production decreased (P<0.05) on d 2 compared with pre-weaning baseline. An increase (P<0.05) in acute phase proteins, fibrinogen and haptoglobin was evident on d 2 to 35 compared with pre-weaning baseline. Concentration of glucose increased on d 2 to 28, whereas non-esterified fatty acid decreased on d 2 to 35 compared with pre-weaning baseline. Post-housing, concentrations of cortisol, rectal body temperature, total leukocyte number, and glucose were unchanged (P>0.05). On d 2 post-housing, neutrophil number and neutrophil: lymphocyte ratio increased (P<0.05), whereas lymphocyte number and concentrations of dehydroepiandrosterone, fibrinogen and non-esterified fatty acid decreased (P<0.05) compared with pre-housing baseline. Concentration of haptoglobin increased (P<0.05) on d 14 to 21 post-housing. Conclusions: A transitory increase in neutrophil number and decrease in lymphocyte number, increased neutrophil:lymphocyte ratio coupled with decreased interferon-gamma production, and increased concentration of acute phase proteins indicate a stress response in cows post-weaning, whereas post-housing, changes were less marked.

Expression levels of uridine 5'-diphospho-glucuronosyltransferase genes in breast tissue from healthy women are associated with mammographic density  (2010-08-27T00:00:00Z)

IntroductionMammographic density (MD), as assessed from film screen mammograms, is determined by the relative content of adipose, connective and epithelial tissue in the female breast. In epidemiological studies, a high percentage of MD confers a four to six fold risk elevation of developing breast cancer, even after adjustment for other known breast cancer risk factors. However, the biologic correlates of density are little known. Methods: Gene expression analysis using whole genome arrays was performed on breast biopsies from 143 women; 79 women with no malignancy (healthy women) and 64 newly diagnosed breast cancer patients, both included from mammographic centres. Percent MD was determined using a previously validated, computerized method on scanned mammograms. Significance analysis of microarrays (SAM) was performed to identify genes influencing MD and a linear regression model was used to assess the independent contribution from different variables to MD. Results: SAM-analysis identified 24 genes differentially expressed between samples from breasts with high and low MD. These genes included three uridine 5'-diphospho-glucuronosyltransferase (UGT) genes and the oestrogen receptor gene (ESR1). These genes were down-regulated in samples with high MD compared to those with low MD. The UGT gene products, which are known to inactivate oestrogen metabolites, were also down-regulated in tumour samples compared to samples from healthy individuals. Several single nucleotide polymorphisms (SNPs) in the UGT genes associated with the expression of UGT and other genes in their vicinity were identified. Conclusions: Three UGT enzymes were lower expressed both in breast tissue biopsies from healthy women with high MD and in biopsies from newly diagnosed breast cancers. The association was strongest amongst young women and women using hormonal therapy. UGT2B10 predicts MD independently of age, hormone therapy and parity. Our results indicate that down-regulation of UGT genes in women exposed to female sex hormones is associated with high MD and might increase the risk of breast cancer.

Microarray data analysis in neoadjuvant biomarker studies in ER+ breast cancer (2010-08-20T00:00:00Z)

Microarray data have been widely utilized to discover biomarkers predictive of response to endocrine therapy in ER+ breast cancer. Typically these data have focused on analyses conducted on the diagnostic specimen. However dynamic temporal changes in gene expression associated with treatment may deliver significant improvements to the current generation of predictive models. We present and discuss some statistical issues relevant to the paper by Taylor and colleagues who conducted studies to model the prognostic potential of gene expression changes that occur after endocrine treatment.

The MET oncogene and basal-like breast cancer: another culprit to watch out for?  (2010-08-23T00:00:00Z)

Recent findings suggest the involvement of the MET oncogene, encoding the tyrosine kinase receptor for hepatocyte growth factor, in the onset and progression of basal-like breast carcinoma. The expression profiles of basal-like tumors - but not those of other breast cancer subtypes - are enriched for gene sets that are coordinately over-represented in transcriptional signatures regulated by Met. Consistently, tissue microarray analyses have revealed that Met immunoreactivity is much higher in basal-like cases of human breast cancer than in other tumor types. Finally, mouse models expressing mutationally activated forms of Met develop a high incidence of mammary tumors, some of which exhibit basal characteristics. This review summarizes the current knowledge on the role and activity of Met in basal-like breast cancer, with a special emphasis on the correlation between this tumor subtype and the cellular hierarchy of the normal mammary gland.

Spatial organization acts on cell signaling - how physical force contributes to the development of cancer (2010-08-23T00:00:00Z)

Cells constantly encounter physical forces and respond to neighbors and circulating factors by triggering intracellular signaling cascades that in turn affect their behavior. The mechanisms by which cells transduce mechanical signals to downstream biochemical changes are not well understood. In their work, Salaita and coworkers show that the spatial organization of cell surface receptors is crucial for mechanotransduction. Consequently, force modulation that disrupts the mechano-chemical coupling may represent a critical step in cancerogenesis.

CYP2D6 gene variants: association with breast cancer specific survival in a cohort of breast cancer patients from the United Kingdom treated with adjuvant tamoxifen (2010-08-23T00:00:00Z)

IntroductionTamoxifen is one of the most effective adjuvant breast cancer therapies available. Its metabolism involves the phase I enzyme, cytochrome P4502D6 (CYP2D6), encoded by the highly polymorphic CYP2D6 gene. CYP2D6 variants resulting in poor metabolism of tamoxifen are hypothesised to reduce its efficacy. An FDA-approved pre-treatment CYP2D6 gene testing assay is available. However, evidence from published studies evaluating CYP2D6 variants as predictive factors of tamoxifen efficacy and clinical outcome are conflicting, querying the clinical utility of CYP2D6 testing. We investigated the association of CYP2D6 variants with breast cancer specific survival (BCSS) in breast cancer patients receiving tamoxifen. Methods: This was a population based case-cohort study. We genotyped known functional variants (n=7; minor allele frequency (MAF)>0.01) and single nucleotide polymorphisms (SNPs) (n=5; MAF>0.05) tagging all known common variants (tagSNPs), in CYP2D6 in 6640 DNA samples from patients with invasive breast cancer from the SEARCH (Studies of Epidemiology and Risk factors in Cancer Heredity); 3155 cases had received tamoxifen therapy. There were 312 deaths from breast cancer, in the tamoxifen treated patients, with over 18000 years of cumulative follow-up. The association between genotype and BCSS was evaluated using Cox proportional hazards regression analysis. Results: In tamoxifen treated patients, there was weak evidence that the poor-metaboliser variant, CYP2D6*6, (MAF=0.01), was associated with decreased BCSS (P=0.02; HR=1.95; 95% CI=1.12-3.40). No other variants, including CYP2D6*4 (MAF=0.20), previously reported to be associated with poorer clinical outcomes, were associated with differences in BCSS, in either the tamoxifen or non-tamoxifen groups. Conclusions: CYP2D6*6 may affect BCSS in tamoxifen-treated patients. However, the absence of an association with survival in more frequent variants, including CYP2D6*4, questions the validity of the reported association between CYP2D6 genotype and treatment response in breast cancer. Until larger, prospective studies confirming any associations are available, routine CYP2D6 genetic testing should not be used in the clinical setting.

Morphologic transformation of human breast epithelial cells MCF-10A: dependence on an oxidative microenvironment and estrogen/epidermal growth factor receptors (2010-09-01T00:00:00Z)

Background: MCF-10A, immortalized but non-transformed human breast epithelial cells, are widely used in research examining carcinogenesis. The studies presented here were initiated with the observation that MCF-10A cells left in continuous culture for prolonged periods without re-feeding were prone to the development of transformed foci. We hypothesized that the depletion of labile culture components led to the onset of processes culminating in the observed cell transformation. The purpose of this study was to define the factors which promoted transformation of this cell line. Results: Changes in levels of phenol red (PHR), hydrocortisone (HC), and epidermal growth factor (EGF) with or without estrogen treatment indicated that both oxidative stress- and estrogen receptor alpha (ERalpha)-mediated pathways contribute to cell transformation. Gene array and Western blotting analyses of cells maintained in our laboratory and of those from other sources documented detectable ERalpha and ERbeta (ERbeta) in this ERalpha-negative cataloged cell line. Results also indicate the possibility of a direct association of EGF receptor (EGFR) and ERalpha in these cells as well as the formation and high induction of a novel ternary complex that includes ERbeta (ERalpha/ERbeta/EGFR) in cells grown under conditions facilitating transformation. Conclusions: Our studies resulted in the development of a growth protocol where the effects of chronic, physiologically relevant alterations in the microenvironment on cellular transformation were examined. From our results, we were able to propose a model of transformation within the MCF-10A cell line in which oxidative stress, ER and EGFR play essential roles. Overall, our work indicates that the immediate microenvironment of cells exerts powerful growth cues which ultimately determine their transformation potential.

Tumour suppressor function of MDA-7 / IL-24 in human breast cancer  (2010-08-24T00:00:00Z)

IntroductionMelanoma differentiation associated gene-7 (MDA-7), also known as interleukin (IL)-24, is a tumour suppressor gene associated with differentiation, growth and apoptosis. However, the mechanisms underlying its anti-neoplastic activity, tumour-specificity and efficacy across a spectrum of human cancers have yet to be fully elucidated. In this study, the biological impact of MDA-7 on the behavior of breast cancer (BC) cells is evaluated. Furthermore, mRNA expression of MDA-7 is assessed in a cohort of women with BC and correlated with established pathological parameters and clinical outcome. Methods: The human BC cell line MDA MB-231 was used to evaluate the in-vitro impact of recombinant human (rh)-MDA-7 on cell growth and motility, using a growth assay, wounding assay and electric cell impedance sensing (ECIS). Localisation of MDA-7 in mammary tissues was assessed with standard immuno-histochemical methodology. BC tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription, MDA-7 transcript levels were determined using real-time quantitative PCR. Transcript levels were analyzed against tumour size, grade, oestrogen receptor (ER) status, nodal involvement, TNM stage, Nottingham Prognostic Index (NPI) and clinical outcome over a 10 year follow-up period. Results: Exposure to rh-MDA-7 significantly reduced wound closure rates for human BC cells in-vitro. The ECIS model demonstrated a significantly reduced motility and migration following rh-MDA-7 treatment (p=0.024). Exposure to rh-MDA-7 was only found to exert a marginal effect on growth. Immuno-histochemical staining of human breast tissues revealed substantially greater MDA-7 positivity in normal compared to cancer cells. Significantly lower MDA-7 transcript levels were identified in those predicted to have a poorer prognosis by the NPI (p=0.049) and those with node positive tumours. Significantly lower expression was also noted in tumours from patients who died of BC compared to those who remained disease free (p=0.035). Low levels of MDA-7 were significantly correlated with a shorter disease free survival (mean = 121.7 vs. 140.4 months, p=0.0287) on Kaplan-Meier survival analysis. Conclusion: MDA-7 significantly inhibits the motility and migration of human BC cells in-vitro. MDA-7 expression is substantially reduced in malignant breast tissue and low transcript levels are significantly associated with unfavourable pathological parameters, including nodal positivity; and adverse clinical outcomes including poor prognosis and shorter disease free survival. MDA-7 offers utility as a prognostic marker and potential for future therapeutic strategies.

Antiproliferative effect of D-glucuronyl C5-epimerase in human breast cancer cells (2010-08-19T00:00:00Z)

Background: D-glucuronyl C5-epimerase (GLCE) is one of the key enzymes in the biosynthesis of heparan sulfate proteoglycans. Down-regulation of GLCE expression in human breast tumours suggests a possible involvement of the gene in carcinogenesis. In this study, an effect of GLCE ectopic expression on cell proliferation and viability of breast carcinoma cells MCF7 in vitro and its potential molecular mechanisms were investigated. Results: D-glucuronyl C5-epimerase expression was significantly decreased in MCF7 cells compared to normal human breast tissue. Re-expression of GLCE inhibited proliferative activity of MCF7 cells according to CyQUANT NF Cell Proliferation Assay, while it did not affect their viability in Colony Formation Test. According to Cancer PathFinder RT Profiler PCR Array, antiproliferative effect of GLCE in vitro could be related to the enhanced expression of tumour suppressor genes P53 (+3.3 fold), E2F1 (+3.00 fold), BRCA1 (+3.5 fold), SYK (+8.1 fold) and apoptosis-related genes BCL2 (+4.2 fold) and NFKB1 (+2.6 fold). Also, GLCE re-expression in MCF7 cells considerably changed the expression of some genes involved in angiogenesis (IL8, +4.6 fold; IFNB1, +3.9 fold; TNF, +4.6 fold and TGFB1, -5.7 fold) and invasion/metastasis (SYK, +8.1 fold; NME1, +3.96 fold; S100A4, -4.6 fold). Conclusions: The ability of D-glucuronyl C5-epimerase to suppress proliferation of breast cancer cells MCF7 through the attenuated expression of different key genes involved in cell cycle regulation, angiogenesis and metastasis molecular pathways supports the idea on the involvement of the gene in regulation of breast cancer cell proliferation.

Effects of cellular iron deficiency on the formation of vascular endothelial growth factor and angiogenesis  (2010-08-19T00:00:00Z)

Background: Young women diagnosed with breast cancer are known to have a higher mortality rate from the disease than older patients. Specific risk factors leading to this poorer outcome have not been identified. In the present study, we hypothesized that iron deficiency, a common ailment in young women, contributes to the poor outcome by promoting the hypoxia inducible factor-1alpha(HIF-1alpha) and vascular endothelial growth factor (VEGF) formation. This hypothesis was tested in an in vitro cell culture model system. Results: Human breast cancer MDA-MB-231 cells were transfected with transferrin receptor-1 (TfR1) shRNA to constitutively impair iron uptake. Cellular iron status was determined by a set of iron proteins and angiogenesis was evaluated by levels of VEGF in cells as well as by a mouse xenograft model. Significant decreases in ferritin with concomitant increases in VEGF were observed in TfR1 knockdown MDA-MB-231 cells when compared to the parental cells. TfR1 shRNA transfectants also evoked a stronger angiogenic response after the cells were injected subcutaneously into nude mice. The molecular mechanism appears that cellular iron deficiency elevates VEGF formation by stabilizing HIF-1alpha. This mechanism is also true in human breast cancer MCF-7 and liver cancer HepG2 cells. Conclusions: Cellular iron deficiency increased HIF-1alpha, VEGF, and angiogenesis, suggesting that systemic iron deficiency might play an important part in the tumor angiogenesis and recurrence in this young age group of breast cancer patients.

3D collagen type I matrix inhibits the antimigratory effect of doxorubicin  (2010-08-13T00:00:00Z)

Background: The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D) context which simulates a natural microenvironment. Methods: To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK) and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation. Results: We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D) culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected beta1 integrin expression nor the state of phosphorylation of FAK and RhoA. Conclusion: This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.

DNA and the chromosome - varied targets for chemotherapy (2004-05-24T00:00:00Z)

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.

Imaging genome abnormalities in cancer research (2004-01-13T00:00:00Z)

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.

Microarray analysis of gene expression during the cell cycle (2003-09-19T00:00:00Z)

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.

MG-132, an inhibitor of proteasomes and calpains, induced inhibition of oocyte maturation and aneuploidy in mouse oocytes (2002-10-08T00:00:00Z)

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.

Chromosome loops arising from intrachromosomal tethering of telomeres occur at high frequency in G1 (non-cycling) mitotic cells: Implications for telomere capture (2004-09-29T00:00:00Z)

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.

Interleukin-27 acts on hepatic stellate cells and induces signal transducer and activator of transcription 1-dependent responses (2010-08-19T00:00:00Z)

Background: Interleukin (IL)-27 is a cytokine belonging to the IL-6/IL-12 cytokine family that is secreted by activated macrophages and dendritic cells and which strongly acts on T-cells and cells of the innate immune system. Not much is known about possible effects of IL-27 on other cell types. It signals via the common IL-6-type-cytokine receptor chain gp130 and the IL-27-specific chain WSX-1. We previously described that IL-27 also stimulates hepatoma cells and primary hepatocytes. The aim of this study was to investigate whether IL-27 would also act on hepatic stellate cells (HSC), the second most abundant hepatic cell type, which would demonstrate a more general role of this cytokine in the liver. Results: Using a human HSC line and primary rat HSC we investigated the signalling characteristics of IL-27 in these cells. We show that IL-27 activates signal transducer and activator of transcription (STAT) 1 and to a minor extent STAT3 in a human HSC cell line and that it leads to the induction of STAT1 target genes such as interferon response factor-1, myxovirus resistance A and STAT1 itself. Similarly we find that IL-27 also elicits STAT1-dependent responses in primary rat HSC. Conclusions: We provide the first evidence for a function of IL 27 in HSC and show that its responses resemble Interferon-gamma-like functions in these cells. Our data suggests that IL-27 may play an important role in the context of liver inflammation by acting on the different liver cell types.

Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells (2010-07-16T00:00:00Z)

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O2)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O2). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15umol/L at 1.5% O2 to 196umol/L at normoxic 21% O2. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-1 alpha (HIF-1 alpha) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.

Early beginnings - the emergence of complex signaling systems and cell-to-cell communication (2010-07-12T00:00:00Z)

This Editorial highlights recent work on the beginnings of multicellular cell signaling.

PC-3 prostate carcinoma cells release signal substances that influence the migratory activity of cells in the tumor's microenvironment (2010-07-13T00:00:00Z)

Background: Tumor cells interact with the cells of the microenvironment not only by cell-cell-contacts but also by the release of signal substances. These substances are known to induce tumor vascularization, especially under hypoxic conditions, but are also supposed to provoke other processes such as tumor innervation and inflammatory conditions. Inflammation is mediated by two organ systems, the neuroendocrine system and the immune system. Therefore, we investigated the influence of substances released by PC-3 human prostate carcinoma cells on SH-SY5Y neuroblastoma cells as well as neutrophil granulocytes and cytotoxic T lymphocytes, especially with regard to their migratory activity. Results: PC-3 cells express several cytokines and growth factors including vascular endothelial growth factors, fibroblast growth factors, interleukins and neurotrophic factors. SH-SY5Y cells are impaired in their migratory activity by PC-3 cell culture supernatant, but orientate chemotactically towards the source. Neutrophil granulocytes increase their locomotory activity only in response to cell culture supernantant of hypoxic but not of normoxic PC-3 cells. In contrast, cytotoxic T lymphocytes do not change their migratory activity in response to either culture supernatant, but increase their cytotoxicity, whereas supernatant of normoxic PC-3 cells leads to a stronger increase than that of hypoxic PC-3 cells. Conclusions: PC-3 cells release several signal substances that influence the behavior of the cells in the tumor's microenvironment, whereas no clear pattern towards proinflammatory or immunosuppressive conditions can be seen.

Adenyl cyclases and cAMP in plant signaling - past and present (2010-06-25T00:00:00Z)

In lower eukaryotes and animals 3'-5'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins.

Chk1 suppressed cell death (2010-09-02T00:00:00Z)

The role of Chk1 in the cellular response to DNA replication stress is well established. However recent work indicates a novel role for Chk1 in the suppression of apoptosis following the disruption of DNA replication or DNA damage. This review will consider these findings in the context of known pathways of Chk1 signalling and potential applications of therapies that target Chk1.

A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe (2010-08-27T00:00:00Z)

In fission yeast the intra-S phase and DNA damage checkpoints are activated in response to inhibition of DNA replication or DNA damage, respectively. The intra-S phase checkpoint responds to stalled replication forks leading to the activation of the Cds1 kinase that both delays cell cycle progression and stabilizes DNA replication forks. The DNA damage checkpoint, that operates during the G2 phase of the cell cycle delays mitotic progression through activation of the checkpoint kinase, Chk1. Delay of the cell cycle is believed to be essential to allow time for either replication restart (in S phase) or DNA damage repair (in G2). Previously, our laboratory showed that fission yeast cells deleted for the N-terminal half of DNA polymerase epsilon (Cdc20) are delayed in S phase, but surprisingly require Chk1 rather than Cds1 to maintain cell viability. Several additional DNA replication mutants were then tested for their dependency on Chk1 or Cds1 when grown under semi-permissive temperatures. We discovered that mutants defective in DNA replication initiation are sensitive only to loss of Chk1, whilst mutations that inhibit DNA replication elongation are sensitive to loss of both Cds1 and Chk1. To confirm that the Chk1-sensitive, Cds1-insensitive phenotype (rid phenotype) is specific to mutants defective in DNA replication initiation, we completed a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain cell viability when grown at semi-permissive temperatures. Our screen identified two mutants, rid1-1 and rid2-1, that are defective in Orc1 and Mcm4, respectively. Both mutants show defects in DNA replication initiation consistent with our hypothesis that the rid phenotype is replication initiation specific. In the case of Mcm4, the mutation has been mapped to a highly conserved region of the protein that appears to be required for DNA replication initiation, but not elongation. Therefore, we conclude that the cellular response to inhibition of DNA replication initiation is distinct from blocking DNA replication elongation, and this difference can be exploited to identify mutants specifically defective in DNA replication initiation.

Mechanisms of mono- and poly-ubiquitination: Ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine (2010-08-13T00:00:00Z)

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionary conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or polyubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2s.

A software solution for recording circadian oscillator features in time-lapse live cell microscopy (2010-07-06T00:00:00Z)

Background: Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/ dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. Results: In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Conclusions: Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series.AvailabilityCGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/.

Coupling histone homeostasis to centromere integrity via the ubiquitin-proteasome system (2010-07-07T00:00:00Z)

In many eukaryotes, histone gene expression is regulated in a cell cycle-dependent manner, with a spike pattern at S phase. In fission yeast the GATA-type transcription factor Ams2 is required for transcriptional activation of all the core histone genes during S phase and Ams2 protein levels per se show concomitant periodic patterns. We have recently unveiled the molecular mechanisms underlying Ams2 fluctuation during the cell cycle. We have found that Ams2 stability varies during the cell cycle, and that the ubiquitin-proteasome pathway is responsible for Ams2 instability. Intriguingly, Ams2 proteolysis requires Hsk1-a Cdc7 homologue in fission yeast generally called Dbf4-dependent protein kinase (DDK)-and the SCF ubiquitin ligase containing the substrate receptor Pof3 F-box protein. Here, we discuss why histone synthesis has to occur only during S phase. Our results indicate that excess synthesis of core histones outside S phase results in deleterious effects on cell survival. In particular, functions of the centromere, in which the centromere-specific H3 variant CENP-A usually form centromeric nucleosomes, are greatly compromised. This defect is, at least in part, ascribable to abnormal incorporation of canonical histone H3 into these nucleosomes. Finally, we address the significance and potential implications of our work from an evolutionary point of view.

Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes (Wed, 01 Sep 2010 07:01:00 PDT)
Cell Biology, general, Laboratory Organisms, general, Drosophila, Neuroscience, general, Electrophysiology, Patch Clamping, Protocol 5453

INTRODUCTION

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission, as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. This protocol details some basic methods for manufacturing microelectrodes used for intracellular recording and two-electrode voltage-clamp (TEVC), and loose patch electrodes used for focal recording. In addition, a method is provided for manufacturing homemade suction electrodes used for nerve stimulation.

Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Neuromuscular Junction (Wed, 01 Sep 2010 07:01:00 PDT)
Cell Biology, general, Laboratory Organisms, general, Drosophila, Neuroscience, general, Electrophysiology, Protocol 5453

INTRODUCTION

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission, as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Focal recording is an extracellular method designed for the study of synaptic activity of one or a few synaptic boutons rather than the ensemble activity of all of the boutons, as occurs with intracellular recording or the two-electrode voltage-clamp (TEVC) method. This is a useful technique for investigating the properties of different motor neurons that innervate the same muscle, applying statistical analysis to discrete synaptic events, and investigating the heterogeneity of synaptic release properties among boutons.

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction (Wed, 01 Sep 2010 07:01:00 PDT)
Cell Biology, general, Laboratory Organisms, general, Drosophila, Neuroscience, general, Electrophysiology, Patch Clamping, Protocol 5453

INTRODUCTION

The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic feature of synaptic transmission as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Although intracellular recording is particularly valuable in revealing membrane potential changes, it has several limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body-wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of minis (spontaneous miniature synaptic potentials) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only at low release conditions when nonlinear summation is not a major issue. The voltage-clamp technique can overcome these limitations by using negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval NMJ preparation.

Determination of Gross Chromosomal Rearrangement Rates (Wed, 01 Sep 2010 07:01:00 PDT)
Bioinformatics/Genomics, general, Genetics, general, Yeast Genetics, Genetic Variation, Laboratory Organisms, general, Yeast, Molecular Biology, general, Genome Analysis, Protocol 5492

INTRODUCTION

Cells devote a significant amount of metabolism to maintaining the stability of their genome and to preventing inappropriate chromosomal rearrangements that are characteristic of many cancers. A simple genetic assay using haploid derivatives of the yeast Saccharomyces cerevisiae provides a means to quantitatively measure the rate at which gross chromosomal rearrangements (GCRs) accumulate in different genetic backgrounds. This assay measures the rate of simultaneous inactivation of CAN1 and URA3 markers placed on a nonessential end of a yeast chromosome and in principle can be implemented in any haploid strain. Rearrangements detected with this assay include broken chromosomes healed by de novo telomere additions and a spectrum of inter- and intrachromosomal fusion events. The GCR assay allows for detailed analysis of the contributions of individual genes and different pathways in the suppression of genomic instability.

Fluorescence In Situ Hybridization of Bacterial Cell Suspensions (Wed, 01 Sep 2010 07:01:00 PDT)
Cell Biology, general, Visualization, Bacterial Genetics, High-Throughput Analysis, general, Fluorescence, Fluorescence, general, Laboratory Organisms, general, Bacteria, Molecular Biology, general, Analysis of Gene Expression, general, Microbiology, In Situ Hybridization, Visualization of Gene Expression, Protocol 5493

INTRODUCTION

The use of fluorescence in situ hybridization (FISH) to identify and enumerate specific bacteria within a mixed culture or environmental sample has become a powerful tool in combining microscopy with molecular phylogenetic discrimination. However, processing a large number of samples in parallel can be difficult because the bacterial cells are typically fixed and hybridized on microscope slides rather than processed in solution. In addition, Gram-positive cells and certain environmental samples present a unique challenge to achievement of adequate cell fixation and uniform hybridization for optimal FISH analysis. Here, we describe a protocol for FISH in solution that can be performed entirely in suspension, in a microcentrifuge tube format, prior to microscopy. This protocol can be applied to both Gram-positive and -negative cells, as well as complex microbial assemblages. The method employs a rapid technique for performing multiple hybridizations simultaneously, which may be used to qualitatively assess the presence of specific phylogenetic groups in bacterial cultures or environmental samples, and/or directly quantify fluorescence by fluorometry or flow cytometry.

Adoptive transfer of splenocytes to study cell-mediated immune responses in hepatitis C infection using HCV transgenic mice (2010-08-20T00:00:00Z)

Background: Hepatitis C virus (HCV) is a major cause of chronic hepatitis and a health problem affecting over 170 million people around the world. We previously studied transgenic mice that express HCV Core, Envelope 1 and Envelope 2 proteins predominantly in the liver, resulting in steatosis, liver and lymphoid tumors, and hepatocellular carcinoma. Herein, the immune-mediated cell response to hepatitis C antigens was evaluated by adoptive transfers of carboxyfluorescein succinimidyl ester (CFSE) labelled splenocytes from HCV immunized mice into HCV transgenic mice. Results: In comparison to non-transgenic mice, there was a significant decrease in the percentage of CFSE-labeled CD4+ and CD8+ T cells in transgenic mouse peripheral blood receiving adoptive transfers from immunized donors. Moreover, the percentage of CFSE-labeled CD4+ and CD8+ T cells were significantly higher in the spleen of transgenic and non-transgenic mice when they received splenocytes from non-immunized than from immunized mice. On the other hand, the percentages of CD4+ and CD8+ T cells in the non-transgenic recipient mouse lymph nodes were significantly higher than the transgenic mice when they received the adoptive transfer from immunized donors. Interestingly, livers of transgenic mice that received transfers from immunized mice had a significantly higher percentage of CFSE labeled T cells than livers of non-transgenic mice receiving non-immunized transfers. Conclusions: These results suggest that the T cells from HCV immunized mice recognize the HCV proteins in the liver of the transgenic mouse model and homed to the HCV antigen expression sites. We propose using this model system to study active T cell responses in HCV infection.

An insertion mutation in ABCB4 is associated with gallbladder mucocele formation in dogs (2010-07-03T00:00:00Z)

Background: ABCB4 functions as a phosphatidylcholine translocater, flipping phosphatidylcholine across hepatocyte canalicular membranes into biliary canaliculi. In people, ABCB4 gene mutations are associated with several disease syndromes including intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis (type 3), primary biliary cirrhosis, and cholelithiasis. Hepatobiliary disease, specifically gallbladder mucocele formation, has been recognized with increased frequency in dogs during the past decade. Because Shetland Sheepdogs are considered to be predisposed to gallbladder mucoceles, we initially investigated ABCB4 as a candidate gene for gallbladder mucocele formation in that breed, but included affected dogs of other breeds as well. Results: An insertion (G) mutation in exon 12 of canine ABCB4 (ABCB4 1583_1584G) was found to be significantly associated with hepatobiliary disease in Shetland Sheepdogs specifically (P < 0.0001) as well as other breeds (P < 0.0006). ABCB4 1583_1584G results in a frame shift generating four stop codons that prematurely terminate ABCB4 protein synthesis within exon 12, abolishing over half of the protein including critical ATP and a putative substrate binding site. Conclusions: The finding of a significant association of ABCB4 1583_1584G with gallbladder mucoceles in dogs suggests that this phospholipid flippase may play a role in the pathophysiology of this disorder. Affected dogs may provide a useful model for identifying novel treatment strategies for ABCB4-associated hepatobiliary disease in people.

Daytime food restriction alters liver glycogen, triacylglycerols, and cell size: a histochemical, morphometric, and ultrastructural study (2010-02-23T00:00:00Z)

Background: Temporal restriction of food availability entrains circadian behavioral and physiological rhythms in mammals by resetting peripheral oscillators. This entrainment underlies the activity of a timing system, different from the suprachiasmatic nuclei (SCN), known as the food entrainable oscillator (FEO). So far, the precise anatomical location of the FEO is unknown. The expression of this oscillator is associated with an enhanced arousal prior to the food presentation that is called food anticipatory activity (FAA). We have focused on the study of the role played by the liver as a probable component of the FEO. The aim of this work was to identify metabolic and structural adaptations in the liver during the expression of the FEO, as revealed by histochemical assessment of hepatic glycogen and triacylglycerol contents, morphometry, and ultrastructure in rats under restricted feeding schedules (RFS). Results: RFS promoted a decrease in the liver/body weight ratio prior to food access, a reduction of hepatic water content, an increase in cross-sectional area of the hepatocytes, a moderate reduction in glycogen content, and a striking decrease in triacylglyceride levels. Although these adaptation effects were also observed when the animal displayed FAA, they were reversed upon feeding. Mitochondria observed by electron microscopy showed a notorious opacity in the hepatocytes from rats during FAA (11:00 h). Twenty four hour fasting rats did not show any of the modifications observed in the animals expressing the FEO. Conclusions: Our results demonstrate that FEO expression is associated with modified liver handling of glycogen and triacylglycerides accompanied by morphometric and ultrastructural adaptations in the hepatocytes. Because the cellular changes detected in the liver cannot be attributed to a simple alternation between feeding and fasting conditions, they also strengthen the notion that RFS promotes a rheostatic adjustment in liver physiology during FEO expression.

Keratin 19 marks poor differentiation and a more aggressive behaviour in canine and human hepatocellular tumours (2010-02-18T00:00:00Z)

Background: The expression of Keratin 19 (K19) was reported in a subset of hepatocellular carcinomas (HCCs). K19 positive HCCs are associated with an increased malignancy compared to K19 negative HCCs. No suitable mouse models exist for this subtype of HCC, nor is the incidence of K19 expression in hepatocellular neoplasia in model animals known. Therefore, we compared the occurrence and tumour behaviour of K19 positive hepatocellular neoplasias in dog and man. Results: The expression of hepatocellular differentiation (HepPar-1), biliary/progenitor cell (K7, K19), and malignancy (glypican-3) markers was semi-quantitatively assessed by immunohistochemistry. The histological grade of tumour differentiation was determined according to a modified classification of Edmondson and Steiner; the staging included intrahepatic, lymph node or distant metastases. Four of the 34 canine hepatocellular neoplasias showed K19 positivity (12%), of which two co-expressed K7. K19 positive tumours did not express HepPar-1, despite the histological evidence of a hepatocellular origin. Like in human HCC, all K19 positive hepatocellular neoplasias were glypican-3 positive and histologically poorly differentiated and revealed intra- or extrahepatic metastases whereas K19 negative hepatocellular neoplasias did not. Conclusions: K19 positive hepatocellular neoplasias are highly comparable to man and occur in 12% of canine hepatocellular tumours and are associated with a poorly differentiated histology and aggressive tumour behaviour.

Chlamydia trachomatis growth inhibition and restoration of LDL-receptor level in HepG2 cells treated with mevastatin (2010-01-28T00:00:00Z)

Background: Perihepatitis is rare but consistently occurring extragenital manifestation of untreated Chlamydia trachomatis infection. Despite of possible liver involvement in generalized C. trachomatis infection, the ability of the pathogen to propagate in the hepatic cells and its impact on liver functions is not thoroughly investigated. The effect of mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on C. trachomatis growth in human hepatoma cell line HepG2 has been studied. Bacterial growth was assessed by immunostaining with FITC-labeled monoclonal antibody against chlamydial lipopolysaccharide and by RT-PCR for two chlamydial genetic markers (16S rRNA and euo). Results: Chlamydial inclusion bodies were seen in approximately 50% of hepatocytes at 48 hours in the post infection period. Lysates obtained from infected hepatocytes were positive in the infective progeny test at 48 and especially in 72 hours after infection initiation. It has been shown that chlamydial infection in hepatocytes also leads to the decline of LDL-receptor mRNA which reflects infection multiplicity rate. Additions of mevastatin (1, 20 and 40 uM) 1 hour before inoculation restored and upregulated LDL-receptor mRNA level in a dose-dependent manner. Mevastatin treatment had no effect on internalization of chlamydial particles. However it reduced drastically the number of chlamydial 16S rRNA and euo transcripts as well as overall infection rate in HepG-2 cells. Complete eradication of infection has been seen by immunofluorescent staining at 40 uM mevastatin concentration, when expression level of chlamydial 16S rRNA and euo was undetectable. Lower concentration of mevastatin (20 uM) promoted euo expression level and the appearance of atypically small chlamydial inclusions, while there was a noticeable reduction in the number of infected cells and 16S rRNA transcripts. Conclusions: C. trachomatis can efficiently propagate in hepatocytes affecting transcription rate of some liver-specific genes. Ongoing cholesterol synthesis is essential for chlamydial growth in hepatocytes. Inhibitors of cholesterol biosynthesis can supplement conventional strategy in the management of C. trachomatis infection.

Ectopic corticotropin-releasing hormone (CRH) syndrome from metastatic small cell carcinoma: a case report and review of the literature (2010-08-31T00:00:00Z)

Background: Cushing's Syndrome (CS) which is caused by isolated Corticotropin-releasing hormone (CRH) production, rather than adrenocorticotropin (ACTH) production, is extremely rare. Methods: We describe the clinical presentation, course, laboratory values and pathologic findings of a patient with isolated ectopic CRH causing CS. We review the literature of the types of tumors associated with this unusual syndrome and the behavior of these tumors by endocrine testing. Results: A 56 year old woman presented with clinical and laboratory features consistent with ACTH-dependent CS. Pituitary imaging was normal and cortisol did not suppress with a high dose dexamethasone test, consistent with a diagnosis of ectopic ACTH. CT imaging did not reveal any discrete lung lesions but there was mediastinal and abdominal lymphadenopathy and multiple liver lesions suspicious for metastatic disease. Laboratory testing was positive for elevated serum carcinoembryonic antigen and the neuroendocrine marker chromogranin A. Serum markers of carcinoid, medullary thyroid carcinoma, and pheochromocytoma were in the normal range. Because the primary tumor could not be identified by imaging, biopsy of the presumed metastatic liver lesions was performed. Immunohistochemistry was consistent with a neuroendocrine tumor, specifically small cell carcinoma. Immunostaining for ACTH was negative but was strongly positive for CRH and laboratory testing revealed a plasma CRH of 10 pg/ml (normal 0 to 10 pg/ml) which should have been suppressed in the presence of high cortisol. Conclusions: This case illustrates the importance of considering the ectopic production of CRH in the differential diagnosis for presentations of ACTH-dependent Cushing's Syndrome.

Two cases of breast carcinoma with osteoclastic giant cells: Are the osteoclastic giant cells pro-tumoural differentiation of macrophages?  (2010-08-23T00:00:00Z)

Breast carcinoma with osteoclastic giant cells (OGCs) is characterized by multinucleated OGCs, and usually displays inflammatory hypervascular stroma. OGCs may derive from tumor-associated macrophages, but their nature remains controversial. We report two cases, in which OGCs appear in common microenvironment despite different tumoural histology. A 44-year-old woman (Case 1) had OGCs accompanying invasive ductal carcinoma, and an 83-year-old woman (Case 2)with carcinosarcoma. Immunohistochemically, in both cases, tumoural and non-tumoural cells strongly expressed VEGF and MMP12, which promote macrophage migration and angiogenesis. The Chalkley count on CD-31-stained sections revealed elevated angiogenesis in both cases. The OGCs expressed bone-osteoclast markers (MMP9, TRAP, cathepsin K) and a histiocyte marker (CD68), but not an MHC class II antigen, HLA-DR. The results indicate a pathogenesis: regardless of tumoural histology, OGCs derive from macrophages, likely in response to hypervascular microenvironments with secretion of common cytokines. The OGCs have acquired bone-osteoclast-like characteristics, but lost antigen presentation abilities as an anti-cancer defense. Appearance of OGCs may not be anti-tumoural immunological reactions, but rather pro-tumoural differentiation of macrophage responding to hypervascular microenvironments induced by breast cancer.

Significance of lobular intraepithelial neoplasia at margins of breast conservation specimens: a report of 38 cases and literature review (2010-08-20T00:00:00Z)

Background: Presence of lobular intraepithelial neoplasia (LIN) is not routinely reported as part of margin assessment in breast conservation therapy (BCT) as in ductal carcinoma in situ (DCIS). With new emerging evidence of LIN as possible precursor lesion, the hypothesis is that LIN at the margin may increase the risk of local recurrence with BCT. The aim is to determine whether there is an increase incidence of recurrence when LIN is found at surgical margins on BCT. Methods: We retrospectively reviewed a total of 1,334 BCT at a single institution in a 10 year period. Inclusion criteria are positive margin with LIN from primary BCT containing invasive and/or in situ carcinoma with comparison to the negative control group who had similar diseases with negative margin for LIN. Results: We identified 38 cases (2.8%) with LIN either lobular carcinoma in situ/atypical lobular hyperplasia (LCIS/ALH) at a margin on initial BCT with 36% recurrence rate. Of the 38 cases: 5 (13%) were lost to follow-up, 12 (32%) had no further procedures performed and 21 (55%) had re-excision. Out of 21 patients who had re-excisions, 12 (57%) had residual invasive carcinoma or DCIS, three (14%) had pleomorphic LCIS and 4 (19%) showed residual classic type LCIS. 71% had significant residual disease (local recurrence) and 29% had no residual disease. A negative control group consisted of 38 cases. We found two patients with bone or brain metastasis and one local recurrence. Clinical follow up periods range from 1 to 109 months. Conclusions: LIN found at a margin on BCT showed a significant recurrent ipsilateral disease. Our study supports the view that LIN seen at the margin may play a role in recurrence.

Inflammatory myofibroblastic pseudotumour of the liver in association with gall stones - a rare case report and brief review. (2010-08-18T00:00:00Z)

Inflammatory myofibroblastic pseudotumours of the liver are rare tumour-like lesions that can mimic malignant liver neoplasms. The symptoms and radiological findings of this rare tumour can pose diagnostic difficulties. We describe a 69-year-old gentleman who was admitted to our department with symptoms suggestive of acute cholecystitis. Ultrasonography and computed tomography of the liver raised the possibility of metastatic liver disease. A core biopsy of the liver was performed to confirm the diagnosis of liver metastasis. Unexpectedly it showed no evidence of malignancy but instead revealed an inflammatory myofibroblastic pseudotumour of the liver. This case report highlights the diagnostic dilemma that arose due to the similarity of appearances between the two pathological entities on imaging and this stresses the need for accurate histological diagnosis so as to avoid unnecessary surgical intervention. To the best of our knowledge, only a minority of cases are reported in the literature associating a hepatic inflammatory myofibroblastic pseudotumour with gall stones.

Testicular fusocellular rhabdomyosarcoma as a metastasis of elbow sclerosing rhabdomyosarcoma: A clinicopathologic, immunohistochemical and molecular study of one case (2010-08-11T00:00:00Z)

Sclerosing rhabdomyosarcoma (SRMS) is an infrequent variant of rhabdomyosarcoma characterized by extensive intercellular hyaline fibrosis. We report the case of a 37 year-old male with a 9x6 cm SRMS on the right elbow. Histologically, the tumor showed an abundant extracellular hyaline matrix with extratumoral vascular emboli and microscopic foci of fusocellular embryonal rhabdomyosarcoma (FRMS) separated by a fibrotic band from the sclerosing areas. One year later the patient presented with a right intratesticular tumor of 1.2x0.8 cm, which was reported as pure FRMS. Immunohistochemically, SRMS was positive only for MyoD1 and Vimentin and negative for Myogenin and Desmin. Both the elbow emboli with the extratumoral foci of FRMS and the intratesticular tumor were positive for Myogenin, MyoD1, Vimentin and Desmin. Using fluorescent in situ hybridization (FISH), the SRMS and the FRMS tumor cells of the elbow and the FRMS tumor cells of the testis were found to be negative for FOXO1A translocation in chromosome 13. PCR chimeric transcriptional products PAX3-FKHR and PAX7-FKHR were not found. Six months following testicular resection, the patient died of multiple metastases in the mediastinum, lung and right thigh.

The past is a foreign country (2010-08-27T00:00:00Z)

The scientific worldview of someone starting graduate school today is very different from that of previous generations.

Elucidating the molecular characteristics of organogenesis in human embryos (2010-08-27T00:00:00Z)

A transcriptomic analysis of early human organogenesis reveals the molecular signature of these processes and provides a valuable resource for identifying and comparing crucial regulators of mammalian embryogenesis.

Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates (2010-08-27T00:00:00Z)

Background: Burkholderia thailandensis is a non-pathogenic environmental saprophyte closely related to Burkholderia pseudomallei, the causative agent of the often fatal animal and human disease melioidosis. To study B. thailandensis genomic variation, we profiled 50 isolates using a pan-genome microarray comprising genomic elements from 28 Burkholderia strains and species. Of 39 genomic regions variably present across the B. thailandensis strains, 13 regions corresponded to known genomic islands, while 26 regions were novel. Results: Variant B. thailandensis isolates exhibited isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (B. pseudomallei-like capsular polysaccharide) closely resembling a similar cluster in B. pseudomallei that is essential for virulence in mammals; presence of this cluster was confirmed by whole genome sequencing of a representative variant strain (B. thailandensis E555). Both whole-genome microarray and multi-locus sequence typing analysis revealed that the variant strains formed part of a phylogenetic subgroup distinct from the ancestral B. thailandensis population and were associated with atypical isolation sources when compared to the majority of previously-described B. thailandensis strains. In functional assays, B. thailandensis E555 exhibited several B. pseudomallei-like phenotypes, including colony wrinkling, resistance to human complement binding, and intracellular macrophage survival. However, in murine infection assays, B. thailandensis E555 did not exhibit enhanced virulence relative to other B. thailandensis strains, suggesting that additional factors are required to successfully colonize and infect mammals. Conclusions: The discovery of such novel variant strains demonstrates how unbiased genomic surveys of non-pathogenic isolates can reveal insights on the development and emergence of new pathogenic species.

Transcription, one allele at a time (2010-08-27T00:00:00Z)

A recent study presents a technique allowing one to image transcription from a single gene copy in live cells, and highlights the dynamic nature of transcriptional regulation.

Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites (2010-08-27T00:00:00Z)

MirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.

Primary mucin-producing tumours of the salivary glands: a clinicopathological and morphometric study (2010-08-01T00:00:00-05:00)

Correspondence: Characterization of hepatitis C virus-induced nasal mucosa remodelling (2010-08-01T00:00:00-05:00)

Correspondence: Response to ‘Dysplastic naevi, again’ (2010-08-01T00:00:00-05:00)

Correspondence: Dysplastic naevi, again (2010-08-01T00:00:00-05:00)

Expression of Bcl-2 family proteins and association with clinicopathological characteristics of oral squamous cell carcinoma (2010-08-01T00:00:00-05:00)

Coutinho-Camillo C M, Lourenço S V, Nishimoto I N, Kowalski L P & Soares F A (2010) Histopathology57, 304–316Expression of Bcl-2 family proteins and association with clinicopathological characteristics of oral squamous cell carcinomaAims:  To characterize the expression of proteins that inhibit (Bcl-2, Bcl-x, Bcl-xL, Bcl-2-related protein A1, BAG-1) or promote (Bak, Bax, Bim/Bod, Bim-Long, Bad, Bid, PUMA) apoptosis and determine possible correlations between the expression of these proteins and clinicopathological features of oral squamous cell carcinoma (OSCC).Methods and results:  Two-hundred and twenty-nine cases of OSCC, arranged in a tissue microarray, were immunohistochemically analysed. The results demonstrated that the absence of vascular invasion was associated with increased expression of Bak, Bax, Bcl-xL, Bcl-2-related protein and PUMA. Increased expression of Bim/Bod and BAG-1 was associated with the presence of perineural infiltration. An increase in Bid and Bim-Long expression was associated with moderately to well-differentiated tumours. Increased expression of the Bcl-2-related protein and PUMA was associated with tumours occurring in the floor of mouth and increased expression of PUMA was also associated with recurrence of the tumour. Multivariate Cox analysis demonstrated that PUMA and Bim-Long were independent factors in prognosis of OSCC.Conclusions:  Our results showed the involvement of the Bcl-2 family of proteins in OSCC tumorigenesis and suggest that the expression of apoptotic molecules might be used as a prognostic indicator for OSCC.

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage (2010-05-17T00:00:00Z)

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.

Evolution of a cluster of innate immune genes (beta-defensins) along the ancestral lines of chicken and zebra finch (2010-04-01T00:00:00Z)

Background: Avian beta-defensins (AvBDs) represent a group of innate immune genes with broad antimicrobial activity. Within the chicken genome, previous work identified 14 AvBDs in a cluster on chromosome three. The release of a second bird genome, the zebra finch, allows us to study the comparative evolutionary history of these gene clusters between from two species that shared a common ancestor about 100 million years ago. Results: A phylogenetic analysis of the beta-defensin gene clusters in the chicken and the zebra finch identified several cases of gene duplication and gene loss along their ancestral lines. In the zebra finch genome a cluster of 22 AvBD genes were identified, all located within 125Kbp on chromosome three. Ten of the 22 genes were found to be highly conserved with orthologous genes in the chicken genome. The remaining 12 genes were all located within a cluster of 58 Kbp and are suggested to be a result of recent gene duplication events that occurred after the galliformes- passeriformes split (G-P split) and where no duplications have happened along the chicken line. Within the chicken genome, AvBD6 was found to be a duplication of AvBD7, whereas the gene AvDB14 seems to have been lost along the ancestral line of the zebra finch. The duplicated beta-defensin genes have had a significantly higher accumulation of non-synonymous over synonymous substitutions compared to the genes that have not undergone duplication since the G-P split. The expression patterns of avian beta-defensin genes seem to be well conserved between chicken and zebra finch. Conclusion: The genomic comparisons of the beta-defensins gene clusters of the chicken and zebra finch illuminate the evolutionary history of this gene complex. Along their ancestral lines, several gene duplication events have occurred in the passerine line after the galliformes-passeriformes split giving rise to 12 novel genes compared to a single duplication event in the galliformes line. After the duplication events, the duplicated genes have been subject to a relaxed selection pressure compared to the non-duplicated genes, thus supporting models of evolution by gene duplication.

Coregulation mapping based on individual phenotypic variation in response to virus infection (2010-03-18T00:00:00Z)

Background: Gene coregulation across a population is an important aspect of the considerable variability of the human immune response to virus infection. Methodology to investigate it must rely on a number of ingredients ranging from gene clustering to transcription factor enrichment analysis. Results: We have developed a methodology to investigate the gene to gene correlations for the expression of 34 genes linked to the immune response of Newcastle Disease Virus (NDV) infected conventional dendritic cells (DCs) from 145 human donors. The levels of gene expression showed a large variation across individuals. We generated a map of gene co-expression using pairwise correlation and multidimensional scaling (MDS). The analysis of these data showed that among the 13 genes left after filtering for statistically significant variations, two clusters are formed. We investigated to what extent the observed correlation patterns can be explained by the sharing of transcription factors (TFs) controlling these genes. Our analysis showed that there was a significant positive correlation between MDS distances and TF sharing across all pairs of genes. We applied enrichment analysis to the TFs having binding sites in the promoter regions of those genes. This analysis, after Gene Ontology filtering, indicated the existence of two clusters of genes (CCL5, IFNA1, IFNA2, IFNB1) and (IKBKE, IL6, IRF7, MX1) that were transcriptionally co-regulated. In order to facilitate the use of our methodology by other researchers, we have also developed an interactive coregulation explorer web-based tool called CorEx. It permits the study of MDS and hierarchical clustering of data combined with TF enrichment analysis. We also offer web services that provide programmatic access to MDS, hierarchical clustering and TF enrichment analysis. Conclusions: MDS mapping based on correlation in conjunction with TF enrichment analysis represents a useful computational method to generate predictions underlying gene coregulation across a population.

A novel paradigm for cell and molecule interaction ontology: from the CMM model to IMGT-ONTOLOGY (2010-02-18T00:00:00Z)

Background: Biology is moving fast toward the virtuous circle of other disciplines: from data to quantitative modeling and back to data. Models are usually developed by mathematicians, physicists, and computer scientists to translate qualitative or semi-quantitative biological knowledge into a quantitative approach. To eliminate semantic confusion between biology and other disciplines, it is necessary to have a list of the most important and frequently used concepts coherently defined. Results: We propose a novel paradigm for generating new concepts for an ontology, starting from model rather than developing a database. We apply that approach to generate concepts for cell and molecule interaction starting from an agent based model. This effort provides a solid infrastructure that is useful to overcome the semantic ambiguities that arise between biologists and mathematicians, physicists, and computer scientists, when they interact in a multidisciplinary field. Conclusions: This effort represents the first attempt at linking molecule ontology with cell ontology, in IMGT-ONTOLOGY, the well established ontology in immunogenetics and immunoinformatics, and a paradigm for life science biology. With the increasing use of models in biology and medicine, the need to link different levels, from molecules to cells to tissues and organs, is increasingly important.

Expression Analysis of G Protein-Coupled Receptors in Mouse Macrophages (2008-04-29T00:00:00Z)

Background: Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results: Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion: The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.

Endothelial adherens junctions and the actin cytoskeleton: an 'infinity net'? (2010-04-08T00:00:00Z)

A recent paper in BMC Biology reports that actin stress fibers in adjacent cultured endothelial cells are linked through adherens junctions. This organization might provide a super-cellular network that could enable coordinated signaling and structural responses in endothelia.See research article http://www.biomedcentral.com/1741-7007/8/11

Genome of a songbird unveiled (2010-04-01T00:00:00Z)

An international collaborative effort has recently uncovered the genome of the zebra finch, a songbird model that has provided unique insights into an array of biological phenomena.See research articles http://www.biomedcentral.com/1471-2164/9/131, http://www.biomedcentral.com/1471-2164/11/220/, http://www.biomedcentral.com/1471-2202/11/46/ and http://www.biomedcentral.com/1741-7007/8/28/

Robust and specific inhibition of microRNAs in Caenorhabditis elegans (2010-04-01T00:00:00Z)

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of numerous target genes. Yet, while hundreds of miRNAs have been identified, little is known about their functions. In a recent report published in Silence, Zheng and colleagues demonstrate a technique for robust and specific knockdown of miRNA expression in Caenorhabditis elegans using modified antisense oligonucleotides, which could be utilized as a powerful tool for the study of regulation and function of miRNAs in vivo.See research article http://www.silencejournal.com/content/1/1/9

The mathematics of sexual attraction (2010-03-29T00:00:00Z)

Pollen tubes follow attractants secreted by the ovules. In a recent paper in BMC Plant Biology, Stewman and colleagues have quantified the parameters of this attraction and used them to calibrate a mathematical model that reproduces the process and enables predictions on the nature of the female attractant and the mechanisms of the male response.See research article: http://www.biomedcentral.com/1471-2229/10/32

Evolution underground: shedding light on the diversification of subterranean insects (2010-03-11T00:00:00Z)

A recent study in BMC Evolutionary Biology has reconstructed the molecular phylogeny of a large Mediterranean cave-dwelling beetle clade, revealing an ancient origin and strong geographic structuring. It seems likely that diversification of this clade in the Oligocene was seeded by an ancestor already adapted to subterranean life.See research article http://www.biomedcentral.com/1471-2148/10/29

Corpus Refactoring: a Feasibility Study (2007-09-13T00:00:00Z)

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.

Nano-Bio-Genesis: Tracing the rise of nanotechnology and nanobiotechnology as 'big science' (2007-07-14T00:00:00Z)

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.

Applied information retrieval and multidisciplinary research: new mechanistic hypotheses in Complex Regional Pain Syndrome (2007-05-04T00:00:00Z)

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.

Biological information specialists for biological informatics (2007-02-12T00:00:00Z)

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.

Generalization through similarity: motif discourse in the discovery and elaboration of zinc finger proteins (2007-10-03T00:00:00Z)

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.

Expression of high-molecular-weight cytokeratin (34{beta}E12) is an independent predictor of disease-free survival in patients with triple-negative tumours of the breast (Wed, 11 Aug 2010 01:36:40 PDT)
Breast cancer

One-fifth of breast cancers have the triple-negative phenotype; a good prognostic marker has yet not been described for these tumours. Tumour microarrays from 58 triple-negative patients treated with surgery followed by chemotherapy were analysed for expression of cytokeratin 5/6 (CK5/6), epidermal growth factor receptor (EGFR), vimentin, p63 and cytokeratin 34βE12. The mean patient age was 59.2 years with a follow-up from 39 to 168 months. Clinicopathological variables and survival data were correlated with biomarker expression. The frequency of expression of cytokeratin 5/6, EGFR, vimentin, p63 and 34βE12 was 33%, 65%, 50%, 19% and 85%, respectively. Each of 34βE12, p63, EGFR and T stage significantly correlated with both disease-free survival and overall survival. T stage and 34βE12 were independent predictors of overall survival in a multivariate analysis. Expression of 34βE12 predicts disease-free and overall survival in patients with triple-negative tumours. Additional studies are planned to confirm these initial findings.

TNM and mesorectal excisions (Wed, 11 Aug 2010 01:36:40 PDT)

Quetiapine-associated cholestasis causing lipoprotein-X and pseudohyponatraemia (Wed, 11 Aug 2010 01:36:40 PDT)
Liver disease, Pancreas and biliary tract, Lipid disorders

A case of intrahepatic cholestasis secondary to treatment with quetiapine in combination with lamotrigine and zopiclone, resulting in severe hypercholesterolaemia without overt lactescence of the plasma, is presented. Abundant lipoprotein-X was seen on lipoprotein electrophoresis. The patient was diagnosed and treated for hyponatraemia which was likely factitious and caused by hypercholesterolaemia. Cholestasis and hypercholesterolaemia resolved over a period of several months after the discontinuation of quetiapine.

E355G mutation appearing in a patient with e19a2 chronic myeloid leukaemia resistant to imatinib (Wed, 11 Aug 2010 01:36:40 PDT)

The development of imatinib is a milestone in the treatment of chronic myeloid leukaemia (CML), and its therapeutic effect has been extensively investigated in patients with CML who carry M-bcr and m-bcr BCR–ABL fusion transcripts. However, knowledge about its therapeutic effect on patients with CML who have the rare BCR–ABL fusion transcript e19a2 (µ-bcr) remains sparse. This report describes a patient with Philadelphia-positive chronic myeloid leukaemia with e19a2 rearrangement, in whom E355G mutation had been acquired. The patient was resistant to imatinib treatment based on conventional cytogenetic and fluorescence in situ hybridisation analysis.

Oropharyngeal lesions and cervical lymphadenopathy: syphilis is a differential diagnosis that is still relevant (Wed, 11 Aug 2010 01:36:40 PDT)
Sexual transmitted infections (bacterial), Immunology (including allergy), Clinical diagnostic tests

Background

Syphilis (lues), a chronic infectious disease caused by Treponema pallidum, has been increasing in incidence during the last few years. Therefore, while clinically it is often not suspected, syphilis is increasingly becoming a differential diagnosis in routine pathology.

Aim

To report our experience with five cases of cervical lymphadenopathy and/or oropharyngeal lesions, clinically thought to be lymphomas, lymph node metastases or carcinoma, in which we made the mostly clinically unsuspected diagnosis of syphilis.

Methods

Fine needle aspiration of enlarged cervical lymph nodes was evaluated by cytology and flow cytometry (fluorescence-activated cell sorting analysis), and biopsies were examined by using histology. In addition, all materials were also subjected to immunostaining, silver staining and molecular (PCR) testing.

Results

Fine needle aspiration cytology revealed follicular hyperplasia in two cases and granulomatous lymphadenitis in one case. In three patients, concomitant biopsy of co-existing oropharyngeal lesions revealed histological findings compatible with syphilis. T pallidum was detected in all cytological and histological samples by immunohistochemistry/immunocytochemistry and PCR. Subsequently, a diagnosis of syphilis was confirmed clinically and by serology.

Conclusions

Syphilitic lymphadenitis is still a relevant differential diagnosis of cervical lymphadenopathy, and it is clinically often not suspected. Co-exisiting oropharyngeal lesions should alert the physician to this differential diagnosis; and lesions with compatible morphology should be tested with immunohistochemistry and immunocytochemistry and/or molecular analysis to confirm the diagnosis of syphilis.