XPD, XPB and TTD-A, code for subunits of Transcription Factor (TF)IIH. Interpreting XPD mutations in the context of available biochemical data led us to propose adverse effects on CDK-activating kinase (CAK) subunit of TFIIH and TFIIH-mediated functions as a relevant mechanism in preeclampsia. In order to gain deeper insight into the underlying biologic mechanisms involving TFIIH-mediated functions in placenta, we analyzed NER/transcription and global gene expression profiles of normal and preeclamptic placentas and studied gene regulatory networks.Preeclampsia is a significant cause of maternal and fetal mortality and morbidity worldwide. We previously reported associations between trichothiodystrophy (TTD) nucleotide excision repair (NER) and transcription gene mutations in the fetus and the risk of gestational complications including preeclampsia. TTD NER/transcription genes, XPD and XPB were consistently expressed from 14 to 40 weeks gestation while expression of TTD-A was strongly negatively correlated  with gestational age. Analysis of gene expression patterns of placentas from a case-control study of preeclampsia using Algorithm for Reconstruction of Accurate Cellular Networks (ARACNE) revealed GTF2E1, a component of TFIIE which modulates TFIIH, among major regulators of differentially-expressed genes in preeclampsia. The basal transcription pathway was among the largest dysregulated protein-protein interaction networks in this preeclampsia dataset. Within the basal transcription pathway, significantly down-regulated genes besides GTF2E1 included those coding for the CAK complex of TFIIH, namely CDK7, CCNH, and MNAT1. Analysis of other relevant gene expression and gene regulatory network data also underscored the involvement of transcription pathways and identified JUNB and JUND (components of transcription factor AP-1) as transcription regulators of the network involving the TTD genes, GTF2E1, and selected gene regulators implicated in preeclampsia.We found high expression of TTD NER/transcription genes in normal human placenta, above the mean of their expression in all organs. Our results indicate that TTD NER/transcription genes are expressed in placenta during gestational periods critical to preeclampsia development. Our overall findings suggest that impairment of TFIIH-mediated function in transcription in placenta is a likely mechanism leading to preeclampsia and provide etiologic clues which may be translated into therapeutic and preventive measures. Preeclampsia is a complex disorder affecting about 7% of all pregnancies and a major cause of severe intrauterine growth restriction (IUGR) and preterm birth . Preecla6) recessive disorder  and XPB  in placenta compared to skin and other tissues. XPD expression was also higher in placenta than skin and most other tissues but the difference did not reach statistical significance  with gestational age  . In our GTF2E1, which codes for a component of TFIIE, as the most interconnected gene in the network   revealed0588  revGTF2E1 with 120 significant interactions  and those coding for all three components of the CAK complex of TFIIH, namely CCNH , CDK7  and MNAT1 (which codes for MAT1)   are inferred to be the master regulator(s) in the network defined as gene(s) with the highest number of significant interactions with other genes within the network. In our analysis, the top gene in the network in terms of the largest number of interactions was -6) between GTF2E1 and FLT1 siRNA datasets suggesting similar consequences for inhibition of FLT1 tyrosine protein kinase and inhibition of transcription in the endothelial cells  transcriptome dataset GSE27871) [871 [27] We analyzed the reactome database  to identGTF2E1) and gene regulators   implica) Figure . These a) Figure , highligAlthough singular observations of certain pregnancy complications were noted in individual case reports of TTD patients (as illustrated in our recent genetic epidemiologic study ), gestata and three XP  The reactome dataset  was alsoIn order to infer potential common transcriptional regulatory networks of selected genes and gene regulators implicated in preeclampsia, analysis of the ENCODE Chip Seq derived regulatory network data  was condaReferences for most XPD mutations analyzed in our previous report can be found in Table four of Cleaver et al. [r et al. .The authors declare that they have no competing interests.RM and AD conceived the study and analyzed the data. XA and VN helped with data analysis and interpretation. AK helped with data preparation and analysis. All authors read and approved the final manuscript.2 scale) of three TTD NER/transcription genes in normal human tissue array including placenta in GSE 96 [NER/transcription gene expression profiles in normal human placenta. Panel a. Bar graph depicting expression (relative fluorescence intensity shown on logn GSE 96 . Red barn GSE 96 . CirclesClick here for file


Acromegaly is a rare endocrine disorder with excess growth hormone (GH) production. This disorder has important metabolic effects in insulin resistance and lipolysis. The objective of this study was to explore transcriptional changes induced by GH in adipose tissue.Patients with acromegaly (n=9) or non-functioning pituitary adenoma (n=11) were prospectively observed from March 2011 to June 2012. The patients underwent clinical and metabolic profiling including assessment of HOMA-IR. Explants of adipose tissue were assayed ex-vivo for lipolysis and ceramide levels. Adipose tissue was analyzed by RNA sequencing (RNAseq).IGF1, IGFBP3) as well as several novel transcriptional changes, some of which may be important for GH signal regulation (PTPN3 and PTPN4) and the effect of GH on growth and proliferation. Several transcripts could potentially be important in GH-induced metabolic changes. Specifically, induction of LPL, ABHD5, ACVR1C could contribute to enhanced lipolysis and may explain the suggestive enhancement of adipose tissue lipolysis in acromegaly patients as reflected by glycerol release from the explants of the two groups of patients (p=0.09). Higher expression of SCD and TCF7L2 could contribute to insulin resistance. Expression of HSD11B1 was reduced and GR was increased, predicting modified glucocorticoid activity in acromegaly.There was evidence of reduced insulin sensitivity based on the increase in fasting glucose, insulin and HOMA-IR score. We observed several previously reported transcriptional changes (We identified the acromegaly gene expression signature in human adipose tissue. The significance of altered expression of specific transcripts will enhance our understanding of the metabolic and proliferative changes associated with acromegaly.


Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion.in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes  Glioblastoma (GBM), the most malignant glioma tumor, is highly invasive with a median survival of 16 months despite combined surgical resection, radiotherapy, and chemotherapy. Although the molecular signature of glioma has been investigated in detail The brain consists of numerous cell types forming tissue scaffolds that guide invasion Glioma stem cells (GSCs) are a subpopulation of cells in the tumor capable of self-renewal that can give rise to a heterogeneous tumor in vitro would affect cell physiology and gene expression. Traditional laser capture microdissection studies of glioma invasion focused on glioma specimens from human patients, dissecting neoplastic astrocytes as the invading cells at the rim of the tumor core Ideally, one would perform between–group differential expression analysis for each cellular compartment. However, experimental methods for isolating the IGCs and their microenvironment cellular compartment are neither timely nor cost effective, and isolating them in situ. Subsequently, we found several genes in multiple pathways that may facilitate invasion which were validated at the protein level in xenografted GSCs and in clinical samples from GBM patients. Importantly, OLIG2, a proliferation regulator and glioma progenitor cell marker was upregulated in the IGCs relative to the tumor core. Inhibition of OLIG2 expression reduced migration and stemness and provided functional confirmation as a potential therapeutic target in glioma invasion. Overall, the novel approach in this study allowed us to construct and evaluate a more complete picture of multiple pathways within the area of glioma invasion than has been previously demonstrated.In the present study we used an innovative laser microdissection (LMD) enrichment strategy and gene expression analysis. Differentially expressed genes (DEGs) of IGCs and their microenvironment were simultaneously detected allowing us to deduce functional networks that characterize glioma invasion Following signed informed consent, tumor tissue was obtained from patients (aged ≥18 years) undergoing medically indicated resection of malignant gliomas at the National Institutes of Health as part of a clinical trial approved by the Institutional Review Board (NCI-02C0140). NCI Animal Care and Use Committee (ACUC) approved all animal experiments.Primary glioma stem cells were cultured in NBE medium as previously described GSCs migration was assessed by the xCELLigence RTCA DP device, according to the manufacturer instructions .An intracranial orthotopic model in SCID mice was utilized for the generation of infiltrative glioma xenograft tumors Raw data for this experiment has been deposited in the Gene Expression Omnibus (accession number GSE53717).The probe for each gene was designed and synthesized by NanoString nCounter technologies to match the Affymetrix probe target region . HousekeThe standard immunohistochemistry of paraffin sections and western blotting were performed as previously described in vitro cell migration assay (P≤0.01). When laminin was used for chemotaxis, only 0923 cells showed significant migration (P≤0.01). In haptotaxis conditions with fibronectin, only 1228A1 cells demonstrated significant migration (P≤0.01). None of the GSCs migrated in chemotaxis with fibronectin. Intracranial tumors generated by 0923 and 1228A1 GSCs demonstrated an extensive infiltration into the surrounding cerebral cortex  in glioma invasion  enrichment analysis. GO terms that were overrepresented and had at least a 30% overlap with at least one other category were identified as significant  and 9 genes of cell polarity  were downregulated STAT3, which is downregulated, acts as a hub in the EMT network interacting with all other networks. On the other hand, majority of genes (13 out of 18) associated with neurophysiological process  and in the cell cycle  were upregulated CTNNB1), which plays a central role in the canonical Wnt pathway, acts as a major hub interconnecting with genes and gene products associated with tissue remodeling and wound repair  For every dataset, we focused on a subset of genes associated with significant cellular processes and canonical pathways  SEMA5A-Plxnb3, AQP4-Dag1, and FGF2-Sdc2) Further analysis identified 17 potential protein-protein binding interactions between the IGCs and the microenvironment residing host cells  and PDGFA proteins in the invasive glioma cells was confirmed (Pdgfra was only detected in host cells residing at the AOI (Shh) protein was only observed at the brain cortex and barely detected at the AOI or tumor core  transcription factor, is selectively expressed in a subgroup of glioma cells and required for glioma formation in a murine GBM model OLIG2 expression by the IGCs in situ may suggest their stemness, which supports their invasive capability in vivo. We thus investigated whether OLIG2 could serve as a therapeutic target in invading glioma cells. We examined the effect of OLIG2 knockdown (shOLIG2) on the differentiation state, self-renewal, proliferation, cell migration and in vivo invasion of GSCs. As demonstrated in NANOG, OCT4 and SOX2 in 0923 shOLIG2 GSCs were significantly decreased compared to the shControl cells. Immunofluorescence labeling  as previously demonstrated GFAP and TUJ1 in 0923 shOLIG2 cells in a more pronounced manner than in shControl cells  and SOX9. Our analysis revealed . These probes were designed to only capture human or mouse genes, and a significant (70–80%) proportion was indeed validated, and is in line with previous reports using pure samples Previous studies of global gene expression in GBM invasion used LCM of patient specimens did not distinguish changes in the microenvironment at the site of invasion CDK1, BUB1, FBX05, ADAM23 and PLXNA4, associated with cell cycle, cell adhesion or neurophysiological processes were found in both datasets. Though upregulated genes within the cell cycle functional category can imply activation of cellular proliferation, we observed the presence of inhibitory proteins of APC such as BUB1 and FBX05, which could promote cell cycle arrest PLXNA4, a semaphorin receptor required for guidance of newly differentiated neurons, was also common to both datasets Many of the DEGs common for both xenografted GSCs were at a low fold-change, but were nonetheless statistically significant. We then looked at the intersection of our DEGs with those previously reported in the invasive rim of 4 human GBMs in an attempt to correlate our xenograft data to a closely related one derived in a clinical setting EPHA2 and EPHA3 in maintaining the undifferentiated state of GSC has been reported EFNB3 enrichment at the AOI accompanied by EPHA4 expression in the IGCs. Cell contact-mediated bidirectional EFNB3/EPHA4 signaling and migration of interneurons during cerebral cortex development has been previously reported, suggesting the possible usage of this interaction during glioma invasion EFNB3 in the cerebral cortex may provide the switch between cell-cell adhesion and cell migration by A-Disintegrin-And-Metalloproteases (ADAM)-mediated shedding ADAM10, ADAM15 and ADAM19 at the AOI.Being exploratory and hypothesis generating, our analysis suggests some potential protein-protein interactions that have never been explored between glioma cells and host cells at the AOI. Recently the role of OLIG2 positive tumor cells within the AOI strongly suggests that the invasive niche contains GSCs OLIG2 in 0923 GSCs decreased the expression levels of their NSC markers , and reduced their self-renewal and proliferation abilities. Exposure of these cells to differentiation conditions reveals the potential of the shOLIG2 GSCs to differentiate more profoundly into distinct populations of glial and neuronal lineages than GSCs which express OLIG2. Moreover, OLIG2 knockdown inhibited their migration potential in vitro and in vivo. Recently, Siebzehnrubl et al. SOX2 and OLIG2 are induced by ZEB1-miR-200 feedback loop in GBM. Our data support their model, as non-invasive cells in the tumor mass express relatively low levels of OLIG2. Our in vitro data further show that OLIG2 can modulate TWIST and SNAI2 protein expression, the central regulators of EMT cancer invasion OLIG2, TWIST and SNAI2 expression within the AOI of xenografted 0923 GSCs.The presence of CHI3L1 (YKL40), CD44, and STAT3in situ contains both invasive and post-invasive (colonizing) glioma cell populations. We postulate that as cells exit the tumor mass and infiltrate brain parenchyma, OLIG2 expression is enhanced, and as they colonize brain parenchyma, expression of EMT markers is diminished.In epithelial cancer, EMT is evoked during tumor cell invasion and metastasis, leading to the generation of cancer cells with stem cell-like characteristics. Metastases are accompanied by a re-differentiation and a MET type transition, both of which are proposed to be a driving force of metastasis, suggesting that MET allows for growth and colonization of the invasive cell in situ. This study revealed several distinct glioma pathways as well as many previously characterized developmental and neurophysiological processes that had never been described in glioma. These multiple pathways serve as a rich reservoir for therapeutic targets that warrant further investigation. Firstly, our data shows that direct targeting of proteins such as CD44CHI3L1PARD3 may be ineffective at this stage of disease as they are already downregulated in glioma cells at the AOI. On the other hand, the identification of molecules selectively expressed by invasive glioma cells such as OLIG2 may allow the development of therapeutic strategies that specifically target this population of cells EFNB3 may be equally amenable to therapeutic targeting.While our approach of filtering out cross-hybridizing genes may have resulted in the loss of some differentially expressed genes, the exploratory nature of this study enabled us to capture for the first time multiple pathways that take place at the junction of tumor invasion and microenvironment Specific therapeutic targeting of glioma invasion is a field in its infancy. Within the tumor-bearing CNS, complex, dynamic and synchronized interactions between glioma tumor cells and tumor-microenvironment residing cells  may sustain glioma cell invasion. Recently, Sottoriva et al. detected the multiple coexisting cell lineages and expression subtypes at the individual GBM patient. They also suggested that these IGCs are a heterogeneous population of malignant cells that survived treatment Figure S1Migration in vitro and in vivo of 0827A2 GSCs. (A) Migration activity of 0827A2 GSCs was tested using the xCELLigence RTCA system. The bars represent the average cell indices at 13 h for the indicated conditions of at least 3 experiments. Asterisks indicate significant  differences in the migration of the cells compared to the control [cells in NBE medium in both upper and lower chambers (NBE/NBE) in uncoated well (−/−)] as determined by t-test. Error bars indicate standard error of the means. (B) Migratory nature of xenografted 0827A2 GSCs. H&E stained section depicting restricted infiltration of GBM cells. Inset depicts 10× magnification, representative infiltration area.  Unsupervised hierarchal clustering in (C) human and (D) mouse arrays of 0827A2 GSCs xenografts shows that the regions are not completely segregated while a clean separation was observed in human and mouse arrays of both 0923 and 1228A1 GSCs xenografts (demonstrated in (TIF)Click here for additional data file.Figure S2Gene ontology and pathway analysis of host cells at the area of invasion (tumor microenvironment). (A) Gene ontology analysis of the microenvironment dataset. Please see legend of (TIF)Click here for additional data file.Figure S3Distinguishing invading human GSCs from mouse cells. Frozen section of xenograft glioma derived from intracranial injection of 1228A1 GSCs were stained with OLIG2 (red), hNuclei (green) and DAPI (blue). Invading GSCs were distinguished by either nuclear size or human-nuclear staining (white arrows). Magnification ×40.(TIF)Click here for additional data file.Figure S4OLIG2, PARD3 and Efnb3 expression in xenografted 1228A1 GSCs. Frozen sections of xenograft glioma derived from intracranial injection of 1228A1 GSC were stained with (A) OLIG2, (B) PARD3 or (C) Efnb3 . DNA was stained with DAPI (blue). At the left side of each panel: intracranial tumor histology  and when available a whole brain tile at same scale. Solid line box (tumor core) and dashed line box (area of invasion) identify magnified (×40) images on right as indicated.(TIF)Click here for additional data file.Figure S5EPHA4, WISP1 and Shh expression in xenografted GSCs. Frozen sections of xenograft gliomas derived from intracranial injection of 0923 (Upper panel) or 1228A1 (Lower panel) GSCs were stained with (A) EPHA4, (B) WISP1 or (C) Shh . Please see legend of (TIF)Click here for additional data file.Figure S6Downregulated expression of EMT associated genes in invasive glioma cells of xenografted 1228A1 GSCs. Frozen sections of xenograft glioma derived from intracranial injection of 0923 GSC were stained with (A) CD44, (B) CHI3L1 or (C) ITGA6 . Please see legend of (TIF)Click here for additional data file.Figure S7PDGFA and mPdgfra expression in xenografted GSC. Frozen sections of xenograft glioma derived from intracranial injection of 0923 (Upper panel) or 1228A1 (Lower panel) GSC were stained with (A) PDGFA and (B) mPdgfra (both in red). Please see legend of (TIF)Click here for additional data file.Table S1NanoString Codeset Details.(XLS)Click here for additional data file.File S1Table S2: Pathway Maps in Invading GSCs and the Tumor Microenvironment. Table S3: Potential Protein-Protein Interactions between Invading GSCs and Their Microenvironment Residing Cells. Table S4: Differentially expressed genes in invading GSCs (array data). Table S5: Differentially expressed genes in the tumor microenvironment (array data). Table S6: Glioma invasion-related genes common with previously reported study by Kislin et al. (reference (DOC)Click here for additional data file.


External quality assurance (EQA) systems are essential to ensure accurate diagnosis of TB and drug-resistant TB. The implementation of EQA through organising regular EQA rounds and identification of training needs is one of the key activities of the European TB reference laboratory network (ERLTB-Net). The aim of this study was to analyse the results of the EQA rounds in a systematic manner and to identify potential benefits as well as common problems encountered by the participants.The ERLTB-Net developed seven EQA modules to test laboratories’ proficiency for TB detection and drug susceptibility testing using both conventional and rapid molecular tools. All National TB Reference laboratories in the European Union and European Economic Area (EU/EEA) Member States were invited to participate in the EQA scheme.A total of 32 National TB Reference laboratories participated in six EQA rounds conducted in 2010–2014. The participation rate ranged from 52.9% - 94.1% over different modules and rounds. Overall, laboratories demonstrated very good proficiency proving their ability to diagnose TB and drug-resistant TB with high accuracy in a timely manner. A small number of laboratories encountered problems with identification of specific Non-tuberculous Mycobacteria (NTMs) (N = 5) and drug susceptibility testing to Pyrazinamide, Amikacin, Capreomycin, and Ethambutol (N = 4).The European TB Reference laboratories showed a steady and high level of performance in the six EQA rounds. A network such as ERLTB-Net can be instrumental in developing and implementing EQA and in establishing collaboration between laboratories to improve the diagnosis of TB in the EU/EEA. Tuberculosis (TB) remains an important public health problem globally and within the European Union (EU) despite significant progress made in the past decade. In 2013, 64 844 TB cases were reported in 28 EU and two European Economic Area (EEA) countries, 6% less than in 2012 and reflecting a decrease in 19 countries [Timely and accurate diagnosis of active TB is a prerequisite for any successful TB control programme and an essential part of the action framework to eliminate TB in low-incidence countries . The labTo ensure compliance with existing international standards of laboratory diagnostics , all labThe introduction of new drug susceptibility testing methods, including rapid culture and molecular tools, and genotyping methodologies necessitated the development of novel as well as modification of existing EQA schemes (eg for rapid molecular tools). A series of recent reports revealed specific problems related to a lack of standardisation in second-line drug (SLD) susceptibility testing and multilocus MIRU-VNTR genotyping –15. The A situation analysis of national TB reference functions across the EU  formed tThe aim of this study was to systematically analyse the implementation of the EQA scheme across the network from 2010 to 2014, assess performance of the laboratories, identify common problems and discuss the potential benefits of the scheme for the participants.A total of six EQA rounds were conducted from 2010 to 2014 . All 34 The ERLTB-Net developed seven modules covering various types of laboratory techniques: (1) microscopy; (2) primary isolation; (3) identification; (4) drug susceptibility testing; (5) nucleic acid amplification testing (NAAT) for rapid detection of Mycobacteria; (6) molecular drug susceptibility testing using cultures; and (7) molecular drug susceptibility testing using crude DNA extracts .Mycobacterium species and drug susceptibility profiles) were used. Specimen panels included strains with varying DST profiles (M. tuberculosis suspensions treated with chloroform [M. tuberculosis complex (MTBC) strains  as well as non-tuberculous mycobacteria (NTM)  in various combinations.Artificial sputa (spiked with mycobacteria) were used for preparation of slides (for microscopy) and specimens for primary isolation and rapid NAAT-based detection; all specimens in the latter panels contained equal concentrations of Mycobacteria. For identification and DST, strains with validated characteristics  and  methods used for species identification (Module 3).Reports received from participating laboratories were analysed against reference results as determined by the EQA provider (NRL Germany and INSTAND e.V), and the percentage of correct results in each module was calculated by dividing the number of correct results by the total number of tests in the module. This provided the score for the given laboratory in the given module. If not all tests within a given module were performed , the overall score was adjusted accordingly. Laboratories scoring ≥80% were issued performance certificates for individual modules. Their individual performances were referred to as “good” (scores 80.0…99.9%) and “excellent’(100.0%) and overall performance of laboratories achieving 80% score was collectively termed “good”. Scores under 80% were considered to indicate poor performance for the specific module(s) and no EQA certificate was issued .Error rates for specific methods were calculated as a proportion of incorrect results relative to a total number of individual tests performed by all laboratories across all rounds; for example in Modules 5 and 6 data was analysed by individual drugs. Proportions were compared using the chi-square test using GraphPad Prizm . A p-value < 0.05 was considered as significant.Mycobacterium species identification module in round 1 comprising 94.1% of laboratories within the network (The number of participating laboratories varied across EQA rounds and modules with the largest number of laboratories (N = 32) participating in the  network . These 3Performance characteristics of participating laboratories in the EQA rounds 1–6 are summarised in The proportion of laboratories using Ziehl-Neelsen (ZN) staining for smear microscopy (Module 1) varied from 46.0% to 58.0% with the remaining laboratories using primarily auramine fluorescent staining; a small number of laboratories (varying from 1 to 4 in different rounds) used the Kinyoun method.There were minor variations in the numbers of laboratories participating in the proficiency testing for microscopy in different rounds with the largest number (N = 27) taking part in round 5 and the smallest (N = 23) in rounds 2 and 4. In rounds 2–6, laboratories demonstrated good and excellent performance with only a small number  of laboratories failing to achieve the 80% threshold. The proportion of laboratories that were awarded certificates was 80.8% in round 1 and 96.2% in round 6. There were no significant differences in error rates and overall performance characteristics between laboratories using different smear staining methods  .M. tuberculosis complex from NTM and identify Mycobacteria species using a range of phenotypic and/or molecular tools. The number of participating laboratories varied from 23 (round 3) to 29 (round 1). Overall scores were good and excellent with only one laboratory in rounds 4 and 6 failing to achieve the 80% threshold. The proportion of laboratories reporting results of species identification varied from 64.3% to 95.7% in different years and largely depended on the composition of the EQA panels (MTBC or NTM) as not all laboratories identify the same spectrum of NTM species routinely on primary specimens. Common problems encountered by the laboratories included reporting the presence of mycobacteria in negative specimens , and inability to detect NTM in positive specimens (N = 2).Within module 2, laboratories were requested to detect the presence of mycobacteria in artificial spiked sputum specimens using bacteriological culture, and (if routinely performed) differentiate In module 3, participants performed species identification of mycobacterial cultures using phenotypic or molecular tools or a combination of both based on standard protocols adopted in those laboratories. The number of participating laboratories ranged from 21 (Rounds 2 and 5) to 32 (Round 1).M. tuberculosis complex  with M. bovis caprae and M. bovis BCG being the most problematic species.Within this module, performance varied considerably with a total of three laboratories across all rounds achieving low scores of 20–40%  and one laboratory misidentifying all specimens in round 5. The majority of participating laboratories, however, demonstrated either good or excellent performance. The proportion of laboratories that were awarded certificates varied from 85.2% (round 4) to 100% (round 6). Common problems experienced by the laboratories included misidentification of NTM and misidentification of species within the M. tuberculosis cultures for sensitivity to five first-line drugs (FLD)  and additionally to fluoroquinolones (FQ), capreomycin (CAP), amikacin (AMK), and kanamycin (KAN) (Modules 3–5) or FQ, CAP, and AMK (Modules 2 and 6) using any validated phenotypic method routinely used in the laboratory. All but one laboratory used automated liquid culture-based systems  for DST for second line drugs; for FLD including PZA methods varied and included Bactec MGIT960 as well as the proportion method on solid media and resistance ratio method on semi-solid and liquid media for PZA.In module 4, laboratories were requested to test five (modules 1–2 and 5–6) or ten (modules 3 and 4) The number of laboratories taking part in module 4 varied from 22 (Rounds 5 and 6) to 31 (Round 1) and overall performance was good with only two laboratories failing to achieve 80% in one round (round 2). Only susceptibility to rifampicin (RIF) and isoniazid (INH) was tested by all participating laboratories; other drugs were tested by a smaller number of laboratories in accordance with their routine practices. Importantly, the proportion of laboratories routinely testing for susceptibility to pyrazinamide (PZA), FQ and AMK increased over the five years from 65.0%, 56.6%, and 52.2% to 100.0%, 77.3%, and 77.3%, respectively, confirming the ability of many laboratories to detect extensively drug-resistant (XDR) strains.Error rates varied substantially between drugs . PZA wasThis module was designed to test proficiency of laboratories in rapidly detecting MTB complex bacteria in sputum specimens using nucleic acid amplification tests (NAAT). The number of participating laboratories ranged from 19 (Round 4) to 22 (remaining rounds) and performance was good with all but two laboratories (in Rounds 5 and 6) achieving scores >80%. The proportion of laboratories using GeneXpert  methodology for TB detection in primary specimens increased from 35.0% in 2011 to 65.2% in 2014. Other methods used for TB detection in primary specimens included BD ProbeTec DTB  used by up to 13.6% of laboratories in different years, and Cobas TaqMan  GT Mycobacteria Direct , and Artus PCR kit  .There were no differences in error rates and overall performance characteristics between GeneXpert and other NAAT methods for rapid MTB complex bacteria identification.In Module 6, proficiency of laboratories in rapidly detecting resistance to selected first  and second-line drugs  on cultures was tested. The number of participants remained relatively stable over five years and ranged from 18 (Round 1) to 22 (Round 2). Nearly all laboratories used line probe assays (LPAs) for molecular DST. A small number of laboratories (<10%) used in-house validated assays including targeted gene sequencing and pyrosequencing,Performance was good with only one laboratory in round 6 failing to achieve the 80% threshold score. The proportion of laboratories adopting technologies for rapid DST for second-line drugs increased from 66.7% (round 2) to 85.0% (round 6). Overall concordance with reference results was high, exceeding 99% for all drugs . Unlike M. tuberculosis strains, and therefore inactivated non-hazardous crude DNA extracts isolated from fully sensitive, poly- and multidrug-resistant strains were used. Module 7 panels were only included in rounds 3 and 4 and results were obtained from 26 and 24 laboratories, respectively.The primary aim of this module was to test the proficiency of the laboratories in rapid detection of highly drug-resistant Overall performance was good with a total of 8 errors reported . All laboratories achieved scores >80%. Errors were not confined to any specific drug and were lower for RIF (0.3%) and higher for INH (1.0%) . ConcordThe primary aim of this study was to systematically analyse the performance of national TB reference laboratories for detection, identification and drug susceptibility testing for TB diagnosis across the EU/EEA through a comprehensive series of EQA rounds, identify common problems and discuss the potential benefits of the EQA scheme developed within the EU-wide TB reference laboratory network.Overall, laboratories demonstrated good proficiency with scores consistently exceeding the 80% threshold in the vast majority of the laboratories. Steady high performance in both phenotypic and rapid molecular methods over five years with only 27 individual module result failures  shows an ability of national TB Reference laboratories to diagnose TB and drug resistance TB with high accuracy in a timely manner. The principal results of our study were in agreement with another report  demonstrChallenges with microscopy and species identification in rounds 1–3 have been addressed through corrective actions including additional training and laboratory visits. No major problems were detected in primary isolation (module 2). An in-depth analysis of laboratory performance for phenotypic DST has allowed us to identify methodological problems with specific anti-TB drugs including PZA, EMB and, interestingly, KAN and AMK. This is largely in agreement with earlier reports demonstrating a lack of standardised methodologies and drug concentrations for PZA (type of media) and KAN  ,19,20 anProficiency of laboratories in identification and DST using rapid molecular tools proved to be good and excellent. High scores were achieved by the laboratories in molecular DST on cultures and crude extracts and there was a significant increase in the number of laboratories adopting molecular techniques (particularly the GeneXpert system), which is critical for the timely detection of MDR strains and prevention of transmission ,5.Our study identified several common challenges in EQA schemes with relatively low participation rates being one of them. Significant variability of participation rates (52.9–94.1%) across modules and/or EQA rounds can be in part explained by the fact that not all laboratories perform all the tests routinely ; data collected outside the current study suggests that up to 25% of NRLs are not routinely engaged in primary laboratory work (unpublished data). A small number of laboratories encountered challenges in sending the results within the agreed timeframe or were unable to take part due to financial constraints . This highlights the importance of adequate management and careful financial planning including allocation of funds for participation in EQA schemes. Other problems encountered in individual laboratories preventing them achieving the 80% threshold included clerical errors (mislabelling etc), incorrect interpretation of molecular and phenotypic tests results, as well as cross-contamination issues. These problems may be indicative of more general problems in the laboratory practice and should be taken into account in the laboratory quality management.Laboratory services play a crucial role in improving the delivery of health care and reducing the prevalence of TB and drug-resistant TB in particular. Quality assessment through proficiency testing is a fundamental tool used to ensure accuracy of test results by comparing quality between laboratories, evaluating performance and detecting errors so that corrective actions, including additional training and support visits, can be taken in a timely manner to restore the high quality of services.M.tuberculosis genotyping [Overall, results of our study and earlier reports on proficiency testing and EQA schemes for notyping ,14 demonParticipating laboratories benefited from the developed EQA program in a number of ways including free of charge EQA and performance certificates essential for their accreditation by relevant national bodies, availability of training for their staff members as well as targeted actions including direct consultations with the EQA providers and support visits from scientists within the network who were able to provide additional expertise and help to NRLs where needed. Steadily high performance during the implementation period suggests that aforementioned network activities addressing the challenges identified through the analysis of EQA results were effective. Sustainability of EQA and training activities at EU/EEA level as well as their further development through inclusion of novel validated methodologies (eg Next Generation Sequencing) are crucial for ensuring timely and high-quality laboratory diagnosis of TB in the EU/EEA.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Cerebral near-infrared spectroscopy (NIRS) represents an exciting prospect for the non-invasive monitoring of cerebral tissue oxygenation in traumatic brain injury (TBI). Earlier attempts at clinical application of cerebral NIRS demonstrated that further work was needed . The bas® computational light modelling software (developed at the University of Birmingham) [The optimal depth for grey matter target tissue (grey-white matter junction) was ascertained by reviewing a series of brain CT scans of patients who had sustained a TBI. Set (average) measurements were derived from these identifying the target depth of the grey matter strip from the point of probe placement. Currently there are five commercially available cerebral NIRS systems. Table mingham) . The novWe reviewed 32 trauma series CT brain images and the average depth to grey matter was derived as 21.37 mm  from the surface (Figure Based on the computational modelling of our work, the FORE-SIGHT NIRS device by CAS Medical source detector layout provides the greatest sensitivity at depth for the purposes of cortical monitoring in trauma. Variations in the layout have a significant impact on the quality of signal detected.


IL1RAPL1, PIR, GABRQ, GPRASP2, SYTL4) with cadherin, protocadherin and ankyrin repeat gene families most commonly altered . Other genes related to neurogenesis and neuronal migration , were also identified.Classical autism or autistic disorder belongs to a group of genetically heterogeneous conditions known as Autism Spectrum Disorders (ASD). Heritability is estimated as high as 90% for ASD with a recently reported compilation of 629 clinically relevant candidate and known genes. We chose to undertake a descriptive next generation whole exome sequencing case study of 30 well-characterized Caucasian females with autism  from multiplex families. Genomic DNA was used for whole exome sequencing via paired-end next generation sequencing approach and X chromosome inactivation status. The list of putative disease causing genes was developed from primary selection criteria using machine learning-derived classification score and other predictive parameters . We narrowed the variant list to 10 to 20 genes and screened for biological significance including neural development, function and known neurological disorders. Seventy-eight genes identified met selection criteria ranging from 1 to 9 filtered variants per female. Five females presented with functional variants of X-linked genes ( PTEN tumor suppressor gene ; d1 = peak height of digested DNA from the first allele and u1 = peak height of undigested DNA from the first allele; d2 = peak height of digested DNA from the second allele and u2 = peak height of undigested DNA from the second allele. Highly skewed XCI is defined as >80% calculated ratio for either one of the AR gene alleles in the digested DNA sample. To ensure the reproducibility of XCI results and equal amplification of both alleles, the digestion, PCR amplification, and genotyping were repeated up to three times in several samples.The digestion process preferentially degrades activated (unmethylated) over inactivated (methylated) DNA. Undigested DNA is preferentially amplified and produces larger peak heights. The peak height values for digested DNA is normalized using peak height values for the undigested DNA for each subject. The percentage of XCI for each XCI status for each subject was assigned to one of the three mutually exclusive categories: (i) randomly selected inactivation of either allele from each X chromosome (XCI = 50%:50% to 64%:36%); (ii) moderately skewed inactivation favoring 1 allele of one of the X chromosomes (XCI = 65%:35% to 80%:20%); and (iii) highly skewed inactivation of a single allele representing one X chromosome (XCI > 80%:20%). The relative frequency of random, moderate, and highly skewed XCI categories was determined for the females with autism. The binomial frequency distribution of X-linked gene variants (present or absent) among females with autism spectrum disorder exhibiting moderate and high levels of XCI skewness were approximated using Yates’ chi-squire test and odds ratios were calculated with 95% confidence intervals.The top genes chosen were based on the final selection criteria identified for each female and evaluated for biologic function and whether each gene was previously reported as a clinically relevant candidate or known gene for autism . The X cCCDC64 in subject HI0605) and some novel ] in 77% of the remaining females. In most females, we found more than one gene that could contribute to the causation of autism. This compares with a recent report of molecular findings among 2000 consecutive patients (primarily pediatric age) referred for clinical whole exome sequencing for evaluation of suspected genetic disorders at a large USA medical center in which 25% received a molecular diagnosis. About 60% of the diagnostic mutations were not previously reported [In summary, we shared our experience using a descriptive next generation whole exome sequencing case study approach examining 30 well-characterized Caucasian females with autism between 5 and 16 years of age recruited from multiplex families enrolled at the AGRE for research purposes. Interpretation of DNA findings are limited due to an inability to study other affected and non-affected family members to determine whether the gene variants were de-novo or inherited in origin for correlation with clinical phenotypes and lack of Sanger sequencing confirmation. Using strict selection criteria, four females (13%) were found to possess single gene variants of known candidate genes for ASD with a high likelihood of causality. We also identified multiple plausible candidate genes [some known (e.g., reported . By phen


Left ventricular ejection fraction (LVEF) is the primary risk stratification tool used for therapeutic decision making related to device therapy. However, an influence of right ventricular dysfunction on cardiovascular events is becoming apparent. The incremental prognostic value of right ventricular ejection fraction (RVEF) on future arrhythmic events among those being considered for device therapy has not been well examined. In this study we evaluate the significance of MRI-based measurement of RVEF versus other clinical and MRI-based variables for their ability to predict Sudden Cardiac Death (SCD) or Appropriate ICD therapy.Consecutive patients with cardiomyopathy were evaluated for device candidacy. All patients underwent a standard Late Gadolinium Enhancement (LGE) MRI protocol followed by a blinded core-laboratory based quantification of left ventricle (LV) and right ventricle (RV) volumes, ejection fraction (EF) and total hyperenhancement (HE). Patients were stratified according to the presence or absence of significant RV systolic dysfunction, defined as an RVEF ≤45%. All patients were followed for the occurrence of SCD or appropriate ICD therapy. The secondary outcome was heart failure admission or non-sudden cardiac death. All clinical and MRI-based variables were evaluated for associations with the primary and secondary outcomes with Cox proportional multivariable regression analysis also performed.A total of 318 patients  were followed over a median of 467 days. At the end of follow-up 49 patients (15.4%) suffered a primary outcome (10 SCD and 39 appropriate therapies). Baseline clinical characteristics were similar among those with and without the primary outcome with the exception of prior history of ventricular arrhythmia and ischemic etiology (p < 0.05). Following adjustment for etiology, LVEF, LVEDV, and Total HE, those with an RVEF≤45% were more likely to experience the primary outcome  and the secondary outcome  of interest.Patients with right ventricular dysfunction, defined as RVEF≤45% by cardiac MRI, are at an increased risk of future arrhythmic events. Similarly, these individuals are at an elevated risk of heart failure admission and non-sudden cardiac death.This research was supported by the Imaging in Cardiovascular Therapeutic grant from the Ontario Research Fund and the Canada Foundation of Innovation.


In order to improve the tracking accuracy, model estimation accuracy and quick response of multiple model maneuvering target tracking, the interacting multiple models five degree cubature Kalman filter (IMM5CKF) is proposed in this paper. In the proposed algorithm, the interacting multiple models (IMM) algorithm processes all the models through a Markov Chain to simultaneously enhance the model tracking accuracy of target tracking. Then a five degree cubature Kalman filter (5CKF) evaluates the surface integral by a higher but deterministic odd ordered spherical cubature rule to improve the tracking accuracy and the model switch sensitivity of the IMM algorithm. Finally, the simulation results demonstrate that the proposed algorithm exhibits quick and smooth switching when disposing different maneuver models, and it also performs better than the interacting multiple models cubature Kalman filter (IMMCKF), interacting multiple models unscented Kalman filter (IMMUKF), 5CKF and the optimal mode transition matrix IMM (OMTM-IMM). Bayes filtering algorithms have been broadly used in target tracking systems ,2,3,4, wBlom and Shalom have proposed the interacting multiple model (IMM) algorithm based on a generalized pseudo-random algorithm to decrease the error of single model algorithm, which will improve the quick response and accuracy of target tracking . The IMMIn this paper, the interacting multiple models five degree cubature Kalman filter (IMM5CKF) based on a five degree cubature Kalman filter and IMM algorithm is proposed to improve the tracking accuracy, model estimation accuracy and quick response of target tracking algorithms. The negative weights of 5CKF go to 0 when the system dimensions go to ∞, so 5CKF is more stable than UKF . The simThe five degree cubature Kalman filter is proposed to improve the accuracy of the traditional Cubature Kalman Filter . It choox, P is the covariance of x. The five degree Cubature Kalman Filter includes two steps, time update and measurement update.Supposing state variables (1) FactorizeThe Cholesky decomposition of (2) Evaluate the cubature points:(3) Evaluate the propagated cubature pointsThe sample points are obtained by propagating the cubature points through the state equation as:(4) Estimate the predicted pointsState prediction (5) Estimate the predicted error covariance:(1) Factorize:(2) Evaluate the cubature points:(3) Evaluate the propagated cubature pointsThe sample points are obtained by propagating the cubature points through the observation equation:(4) Estimate the predicted measurement:(5) Estimate the innovation covariance matrix:(6) Estimate the cross-covariance matrix:(7) Estimate the Kalman gain:(8) Estimate the updated state:(9) Estimate the corresponding error covariance:In the paper, the proposed IMM5CKF includes the merits of the 5CKF algorithm and IMM algorithm. The main character of IMM5CKF is that it calculates the state distribution and error covariance matrix by choosing an odd number of special cubature points with equal weights, and the negative weights go to 0 when the dimension of the system goes to The mixed initial value and measure value  of position and velocity are used to contrast the performance of the filtering algorithms. The RMSE defined in state vector The RMSEs of the IMM5CKF, IMMCKF, IMMUKF, 5CKF and OMTM-IMM are shown in In this paper, IMM5CKF is proposed to enhance the tracking accuracy, model estimation accuracy and response sensitivity of nonlinear maneuvering target tracking problems. The algorithm introduces a five degree cubature Kalman filter into interacting multiple models which simultaneously disposes of all the models through a Markov Chain. A classical target tracking problem is utilized to demonstrate that the IMM5CKF can indeed improve the quick response sensitivity of target tracking algorithm, and it exhibits more accurate than IMMCKF, IMMUKF, CKF and OMTM-IMM. In our future research, the study may focus on multisensor navigation and positioning systems. The proposed algorithm should be suitable for the complex real environments according to the analysis.


Left ventricular late gadolinium enhancement (LV-LGE) is a known incidental cardiac magnetic resonance (CMR) finding in atrial fibrillation (AF) patients and has recently been associated with increased mortality . While LA 1:2 observational case-control study was conducted with a dataset of cases with new onset ischemic stroke after 1st CMR scan and controls matched by age and sex. Cases and controls were selected from a database of 762 AF patients without any prior history of MI who underwent cardiac MRI exams between June 2006 and January 2013. We manually reviewed the electronic medical records of all patients with CMR and sequentially selected the first identified age and sex matched controls (subjects without stroke event after their CMR scan). Two experienced readers scrutinized the CMR scans of cases and controls for presence of left ventricular delayed enhancement. We then collected all comorbidities and conducted univariate (Chi-square test/t-test as appropriate) and multivariate analyses to examine the association between LV-LGE and occurrence of stroke.Out of 762 patients, we identified 14 cases (1.8%) with new onset ischemic stroke after the first MRI scan. Of these cases, 2 (14%) were found to have LV-LGE. We age and sex matched these 14 cases with 28 controls in a 1:2 fashion. Description of demographic variables and univariate measures of association are presented in Table In this case-control study of AF patients without a history of MI, an incidental finding of LV-LGE is shown to predict the risk of stroke, especially in the case of patients with a higher CHADS2 score.Not applicable.


Furthermore, our study indicates that the structure of CsA during dissociation from the protein possesses a distribution of conformations different from those in solution under equilibrium condition.The immunosuppressive drug cyclosporin A (CsA) binds to its receptor protein cyclophilin 18 (Cyp18) in two distinct kinetic phases, while the mechanism remains elusive. Stopped-flow measurements coupled with titration and competition experiments were used to investigate the puzzling two-phase process of CsA and Cyp18 interaction. This study leads to the dissection of different conformational fractions of either direct fast binding or slow binding with rate-limiting conformational inter-conversion and the real-time measurement of  Berkeley Modenna method[-1 (The slow binding conformer may need to convert to the active conformer before binding to Cyp18 (Model II). Thus, the rate of slow binding represents the apparent converting rate. When the concentration of CsA is lower than that of Cyp18, the slow binding conformers will bind to Cyp18 once they convert to the active conformation with the rate of kna method was usedethod[-1 . This reOur work provides the evidence that the two-phase binding kinetics of CsA to Cyp18 is caused by the ligand conformational heterogeneity. This leads to the dissection of different conformational fractions of either direct fast binding or slow binding with rate-limiting conformational inter-conversion. Moreover, the competition experiment described in S1 FigTime courses of Cyp18 intrinsic fluorescence intensity upon to the adding of THF(A) or LiCl/THF(B). (A) Time courses of Cyp18 intrinsic fluorescence with (Red) or without (Black) subtraction of reference curve (only THF). The arrows show the procedures of the experiments and the curves before and after subtraction of reference curve. (B) Time courses of Cyp18 intrinsic fluorescence with (Red) or without (Black) subtraction of reference curve (only LiCl/THF). The arrows show the procedures of the experiments and the curves before and after subtraction of reference curve.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig−3 s-1.The values shown in the inset represent the different ratios of CsA to CypA. The k value was 8.25 ± 0.03 x 10(PDF)Click here for additional data file.S4 FigBlack curves are plotted by the data converted from (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Tables-f and kf-s used for fitting was board (highlight), whereas the fitting showed closed value to the predicted, this indicated the conformational selection model was the most dominant process of CsA binding to Cyp18 in aqueous solution.The range of k(PDF)Click here for additional data file.


The aim of our investigation was to determine effect of the internal carotid arteries (ICAs) tortuosity (S-shape), kinking and coiling on the arterial blood flow by magnetic resonance imaging and ultrasound (US).50 healthy volunteers (control group) and 43 patients with carotid abnormalities (CAs) were examined on 1.5T MR-scanner using routine MR protocol and quantitative MR angiography (qMRA) for estimate of the arterial blood flow velocity and cross sectional area of the ICA. We observed the cross sectional area and the values of peak velocity, mean velocity, flux velocity of arterial blood flow at one cardiac cycle at cervical and intracranial segments of the ICA .


Immune reconstitution inflammatory syndrome (IRIS) is a common cause of death and even more common cause of morbidity in patients started on highly active antiretroviral therapy (HAART). At present, very limited data is available about IRIS in the local population so the present study was conducted to determine the incidence, clinical manifestations, risk factors and outcomes of IRIS in south Indian HIV/AIDS patients.Study was a prospective surveillance method to identify IRIS from two ART centers in Mysore city, India for a period of two years. HIV patients who are initiated with HAART were included for the study and were followed up for a period of six months. Data were analyzed using SPSS version 21.s (11%) and other opportunistic infections (27%). The major manifestation of IRIS were fever (44%) followed by lymphadenitis (36%). Statistically important predictors for occurrence of IRIS were male gender and low CD4 count. Two deaths were attributable to IRIS and 54% of them required hospitalization.Out of 798 people followed 82 patients had experienced IRIS with an incidence rate of 10%. Among them 48% were unmasking and rest were paradoxical worsening. Diagnosis included tuberculosis (44%), herpes zoster (18%), cryptococcal meningitiPatients with advanced immunodeficiency at HAART initiation are at greatest risk of developing IRIS and should be appropriately screened and monitored. Educational programs on recognizing and treating these conditions should be initiated in ART access programs.


Infaunal communities of benthic macro-organisms (≥ 1mm length) were studied from 81 samples collected across nine sites to the north and south of the Orange River in the Benguela upwelling ecosystem in 2003, with a view to describing communities and understanding the drivers of regional community structure, as well as to document diversity and to examine geographic affinities. Although the fauna was dominated by polychaetes and peracarid crustaceans, patterns in community structure could only weakly be explained by the measured environment (~35%). This is attributed to the generalist nature of the species recovered, which were widely distributed amongst different sediments, water-depths and latitudes. The fauna is dominated by species that enjoy a widespread regional and global distribution and is characterised by relatively low diversity, which is discussed. Coastal-upwelling ecosystems are amongst the most productive in the world, where pelagic systems are characterised by short diatom-based food chains leading to industrial scale fisheries . Biomass2 l-1), and sometimes high levels of hydrogen sulphide in the pore water [sensu [cf 8]. Macrofaunal diversity within OMZs is also low, and communities show a remarkable homogeneity in composition across wide spatial scales; a fact that is attributed to the lack of biogenic structures and large burrowers ).The results of this study suggest that the fauna of the soft-sediment environment off southern Namibia and off Namqualand is characterised by relatively low diversity and by species with a generalist habit and wide distribution. Further work on measuring a wider suite of environmental variables is desirable, and studies into the detailed taxonomic identity of all material collected is encouraged.S1 Fig(TIF)Click here for additional data file.S2 FigNumber  of identified species recovered in one or more of the nine regions (a) or sediment textural groups (b) sampled off southern Namibia and off Namaqualand during 2003.(PPTX)Click here for additional data file.S1 TableList of the identified species recovered from the samples collected off southern Namibia and off Namaqualand during 2003, with an indication of their presence (1) or absence (0) in on-line distributional databases . Information from other sources also indicated, where appropriate. A summary of the known distribution of each species is also indicated.(DOCX)Click here for additional data file.S2 TableList of additional genera, not included in (DOCX)Click here for additional data file.S3 TableMean abundance and standard error (per sample) of the top ten most commonly represented families at each of the sites sampled off southwestern Africa during 2003.(DOCX)Click here for additional data file.S4 TableAverage (root-root) abundance per sample of those macro-infaunal taxa identified by the SIMPER routine in PRIMER 6 as being responsible for 90% of the identity of each of the water-depth zones . The weighted mean water-depth (m) occupied by each of the identified species is also shown.(DOCX)Click here for additional data file.S5 TableAverage (root-root) abundance per sample of those macro-infaunal taxa identified by the SIMPER routine in PRIMER 6 as being responsible for 90% of the identity of each of the sediment textural groups identified by Folk (1954) sampled off southwestern Africa during 2003. The weighted mean sediment particle size (μm) occupied by each of the identified species is also shown.(DOCX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Focal left atrial tachycardia (FLAT) although a common cause of supraventricular tachycardia(SVT) among children, the one's arising from left atrial appendage (LAA) present a unique challenge for successful ablation because of anatomical location. We present two children with FLAT arising from the epicardial LAA, successfully mapped and ablated through percutaneuous epicardial approach. The LAA is one of the major sources of FLATs in children . HoweverA 12 year old girl presented with history of recurrent episodes of palpitations and fatigue. She had left ventricular dysfunction secondary to (TIC). Tachycardia was refractory to multiple antiarrhythmic drugs and was referred for RFA. Patient was in incessant atrial tachycardia and baseline 12 lead electrocardiogram (ECG) revealed negative P waves in leads I and avL, positive P wave in leads V1, III and avF and isoelectric P wave in lead II . ActivatThe second case was a 6 year old boy presented to us with incessant atrial tachycardia at the rate of 190 per minute with 1:1 AV conduction and TIC. The 12 lead ECG showed negative P waves in leads I and avL and positive P waves in the inferior leads and in lead V1 . He had There have been several case series of successful catheter ablation of an LAA tachycardia using different approaches -6. To thThus, the epicardial catheter ablation may be an alternative approach to endocardial catheter ablation in atrial tachycardias with an LAA origin without the need for subjecting these patients to surgical ablation.


Recent reports suggest that within the heterogeneous population that constitutes a melanoma, certain cell types exhibit molecular and functional features similar to stem cells. These melanoma-initiating cells (MICs) have the ability of unlimited self-renewal, multilineage differentiation and the potential to initiate and maintain tumor growth . FurtherMICs were enriched by Fluorescence Activated Cell Sorting using the metabolic marker Aldehyde Dehydrogenase (ALDH) and by establishing melanoma spheres in non-adherent culture conditions from 20 human melanomas. Expression of SOX2 and of HH pathway components was determined by real time PCR and Western blot analysis. Modulation of the HH signaling was performed by stable expression of lentiviral vectors encoding short-interference RNAs specific for SMO or GLI1 (to inhibit HH), and PTCH1 (to activate HH). Functional analysis of SOX2 was performed by stable overexpression and silencing using lentiviral vectors.SOX2 promoter in melanoma cells. Functionally, we show that SOX2 is required for HH-induced melanoma cell growth and MIC self-renewal. We present evidence that SOX2 is highly expressed in a population enriched for cancer stem cells in patient-derived melanomas. Knock-down of SOX2 sharply decreases self-renewal of melanoma spheres and of ALDHhigh melanoma stem cells. Consistently, ectopic expression of SOX2 in melanoma cells is sufficient to enhance stemness in vitro. SOX2 silencing also inhibits cell growth and induces apoptosis in melanoma cells. Most importantly, depletion of SOX2 drastically impairs tumorigenicity of ALDHhigh MICs in orthotopic xenografts.We find that the HH signaling regulates the expression of SOX2 and the downstream effectors of the HH signaling, the transcription factors GLI1 and GLI2, bind to Our data identify SOX2 as a novel mediator of the HH signaling in melanoma cells and indicate that SOX2 is a critical factor for the survival of MICs. These findings could provide the basis for novel therapeutic strategies based on the inhibition of SOX2 for the treatment of human melanomas.


Reprogramming of metabolic pathways and abnormal acetylation of DNA are two common features of cancer, including prostate cancer (PCa).CWR22Rv1 cells (a castrate-resistant human PCa cell line) were treated with a polyherbal mixture of botanicals (PHBs) consisting of 10 herbal extracts [de novo lipid biosynthesis through the phosphorylation of acetyl-CoA carboxylase and by inhibiting the expression of fatty acid synthase and SREBP1c. Also via AMPK, the PHBs inhibit the activity of mTORC1 complex by phosphorylating raptor, decreasing the activity of S6K. These effects can be mimicked by treatment of AICAR (activator of AMPK) and over-expression of AMPKalpha, and attenuated by compound C (AMPK inhibitor) and siRNA silencing of AMPK. Furthermore, the PHBs increase the cellular pools of acetyl CoA via mechanisms that may include ATP-citrate lyase. Coordinately, there are increases in acetylation of histone 3 and expression of the tumor suppressor proteins p21 and p27, a mechanism that most likely involves the down regulation of class I and II histone deacetylases and up regulation of histone acetyl transferase  via ERK1/2 signaling. The inhibitory effects on cell proliferation can be attributed in part to the synergistic activity of some of the individual components in the PHBs as determined by the Chou-Talalay method [in vitro.Our results show that the PHBs can modify lipogenesis in CWR22Rv1 cells by enhancing the activation of AMPK, with concomitant decreases of Bioactive nutrients derived from multiple herbs inhibit the growth of androgen-deprived castrate resistant PCa cells via multiple mechanisms, one of which involves up regulation of AMPK signaling.


P. falciparum resistance mechanism to ACT has become a global research goal. The selective pressure of ACT and the possibility that some specific Pfcrt and Pfmdr1 alleles are associated with treatment failures was assessed in a clinical trial comparing ASAQ to AL in Nanoro. Dried blood spots collected on Day 0 and on the day of recurrent parasitaemia during the 28-day follow-up were analyzed using the restriction fragments length polymorphism (PCR-RFLP) method to detect single nucleotide polymorphisms (SNPs) in Pfcrt (codon76) and Pfmdr1  genes. Multivariate analysis of the relationship between the presence of Pfcrt and Pfmdr1 alleles and treatment outcome was performed. AL and ASAQ exerted opposite trends in selecting Pfcrt K76T and Pfmdr1-N86Y alleles, raising the potential beneficial effect of using diverse ACT at the same time as first line treatments to reduce the selective pressure by each treatment regimen. No clear association between the presence of Pfcrt and Pfmdr1 alleles carried at baseline and treatment failure was observed.The adoption of Artemisinin based combination therapies (ACT) constitutes a basic strategy for malaria control in sub-Saharan Africa. Moreover, since cases of ACT resistance have been reported in South-East Asia, the need to understand  Plasmodium falciparum resistance to antimalarial drugs is the main challenge for malaria control in endemic countries. To overcome resistance to chloroquine (CQ), artemisinin-based combination therapies (ACT) have been recommended by the World Health Organization (WHO) for the treatment of uncomplicated malaria  to distinguish between recrudescence and new infections constitutes another limit of this study and could partially explain why no significant difference was observed between the two groups (recrudescence and new infections). In this study the prevalence of mutation in Pfmdr1-D1246Y was extremely low and it was difficult to correlate the presence of this mutation and treatment outcomes. No samples carried mutation in Pfmdr1-S1034C and Pfmdr1-N1042D as reported by several studies throughout Africa [Our findings suggest also no association between the presence of s report . The Pfmfantrine . Therefos report . Furthert Africa , 30.Pfcrt K76T and Pfmdr1-N86Y alleles, raising the potential beneficial effect of using diverse ACT at the same time as first line treatments to reduce the selecting pressure by each treatment regimen. No association between the presence of Pfcrt and Pfmdr1 alleles carried at baseline and ASAQ treatment failure was observed.AL and ASAQ exerted opposite trends in selecting S1 Fig(XLSX)Click here for additional data file.


Quantitative real-time reverse transcription-polymerase (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as the cytoprotection or inflammation. The selection of an adequate internal reference gene for accurate and consistent analysis of gene expression is of major importance. Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to the gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of the CO on gene expression in cultured cortical astrocytes. The cells were exposed to CO by incubation with CORM-A1 (CO releasing molecule A1) and four different algorithms  were applied to better evaluate the stability of eight putative reference genes. Our results indicate that Gapdh  and Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha , Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-1 for up to 60 min. Analysis of the effects of CO on the expression of Bdnf and bcl-2 gave different results depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. This study highlights the need for proper and accurate usage of reference genes in quantification of qRT-PCR results in studies on the effect of CO in gene expression.


Permanent neonatal diabetes mellitus (PNDM) is a rare disease which is defined as the onset of diabetes before the age of 6 months with persistence through life. Patients with KCNJ11 or ABCC8 gene mutations have the opportunity to switch to oral sulfonylurea therapy. There were limited studies about the genetic analysis and long term follow-up of PNDM.Report four cases of PNDM, including their genetic mutations, treatments and long-time follow-ups. All of the patients and their parents got gene analysis include INS, KCNJ11 or ABCC8 gene.None of the patients and their parents suffered from any genetic mutations of these three common genes. One of the children got continuous subcutaneous insulin infusion (CSII) and the others got multiple injections of insulin (MII). The PNDM patients had persisted after 35 months to 60 months of follow-up, 3 patients maintained almost stable blood sugar level, and 1 patient had poor sugar control.All of PNDM patients are suggested undergo genetic evaluation. For patients without KCNJ11 and ABCC8 gene mutation, oral sulfonylurea might not be considered. CSII is a useful tool for overcoming the difficulties of diabetes, and can also improve quality of life.


Liocarcinus depurator we assessed the contribution of historical and contemporary processes on population connectivity patterns across three main oceanographic discontinuities along the Atlantic-Mediterranean transition: Gibraltar Strait, Almeria-Oran Front and Ibiza Channel. A partial fragment of the cytochrome oxidase I gene was sequenced in 366 individuals collected from localities at both sides of each discontinuity during three time periods. Although localities showed genetic fluctuations through time, a significant gradient was detected along the coast for all sampling periods. Significant inter-annual differences identified within the Alicante area, north of the Almeria-Oran Front, were associated with shifts in the relative contribution of Atlantic and Mediterranean water masses. The persistence of a clinal pattern in the Atlantic-Mediterranean transition area together with local fluctuations suggests a complex balance of dispersal and selection.Spatial genetic studies often require sampling broadly separated areas, difficult to access simultaneously. Although comparing localities surveyed at different time periods might result in spurious genetic differentiation, there is a general believe on the stability of genetic structure through time, particularly if sampled localities are isolated or very distant. By analysing spatial and temporal genetic differentiation of the portunid crab  Spatial genetic studies have increased in the last decades in many taxa while temporal genetic studies, despite being essential to understand biodiversity distribution, are still scarce. Landscape genetics and phylogeography are both concerned with understanding the distribution of genetic variation across natural environments1358The large population sizes of marine organisms generally result in little genetic differentiation despite restricted gene flow, and molecular markers show practical limitations in making inferences for recent timescales and single time point samples111314Three oceanographic discontinuities along the Atlantic-Mediterranean transition in the south and eastern Iberian Peninsula  have been shown to restrict gene flow in several marine species. The connection of the Mediterranean Sea and the Atlantic Ocean through the Gibraltar Strait is characterized by the inflow of surface Atlantic water and outflow of deeper Mediterranean water1791419121423182512Liocarcinus depurator showed that the Almeria-Oran front did not represent a significant barrier to gene flow, whereas Ibiza Channel acted as the main genetic barrierL. depurator across the three main oceanographic discontinuities along the Atlantic-Mediterranean transition. A partial region of the mitochondrial DNA cytochrome oxidase subunit I (COI) was analysed in 366 individuals from five areas situated at both sides of each discontinuity  and sampled during three time periods , and another including haplotypes mostly found in the Mediterranean (Ldep02 and related haplotypes) and were thus referred as the Atlantic (ATL) and the Mediterranean (MED) haplogroups, respectively  the present study evidences that contemporary processes impact local genetic diversity and modify connectivity between populations. Temporal variability of oceanographic discontinuities should not be overlooked when defining connectivity patterns because this could mislead conservation decisions (e.g. when designing networks of marine protected areas)28Liocarcinus depurator populations under strong influence from Atlantic waters and decaying diversity going into the Mediterranean, suggests an Atlantic origin of the species. Many phylogeographic studies identify genetically differentiated units that can be attributed to vicariance or selection reducing gene flowCOI haplotypes for L. depurator can be related to an abrupt decline of sea temperature in north Atlantic waters around 38–40 kya14L. depurator probably diverged due to vicariance events and posterior range expansions, as observed in other species influenced by climatic fluctuations during the Quaternary3435Changes in environmental conditions  and historical shifts of species distributions can shape genetic variation, as shown by spatially-explicit demographic modelling of genetic differentiationLiocarcinus depurator epipelagic larvae could drive genetic connectivity of this species by moving along with surface currents4et al.The greater diversity observed in samples under stronger Atlantic influence  using microsatellite loci suggesting genetic exchange from the west side of the Almeria-Oran front towards the east and northet al.The largest fluctuations in haplotype frequency across time were found in ALAC located between two main oceanographic discontinuities, the Almeria-Oran front in the south and the Ibiza Channel in the north. The ALAC07 sample showed a relatively high frequency of individuals from the Atlantic (ATL) haplogroup, whereas ALAC09 and ALAC13 were clearly dominated by haplotypes from the Mediterranean (MED) haplogroup. A strong genetic break between Almeria-Oran front and Ibiza Channel and higher proportion of Atlantic genotypes was also observed in the comber  pointed to the effect of variable reproductive success, and highlighted the unpredictable nature of connectivity in coral reef fishes43Clupea arenguset al.et al.Liocarcinus depurator as well as in other species with high dispersal capabilities  gene fragment was amplified using the universal primers LCO1490 and HCO2198et al.2 (25 mM), 0.5 μl dNTP’s (1 mM) and 0.4 μl (10 μM) of each primer. PCR products of 2013 samples were cleaned with ExoSAP (1.2 U Exonuclease and 1.2 U Shrimp Phosphatase per μl) at a proportion of 1:2 (ExoSAP:PCR product) and sequenced at the Scientific and Technical Services of the University of Barcelona (Spain) or at Macrogen Inc. .Muscle tissue from each individual was preserved in pure ethanol on board of the vessel. Total DNA extraction was performed for 2009 and 2010 samples using Chelex 10% as in Estoup ST values and their significance assessed performing 1000 permutations with Arlequin v. 3.5P-values were corrected for multiple comparisons using the false discovery rate (FDR) as in NarumST pairwise distances with GenAlEx v6.5et al.Sequences were visually inspected, aligned and trimmed, to obtain a final alignment of 527 bp, with BioEdit v7.2.5Liocarcinus depurator haplotypes were estimated by Bayesian Inference (BI) using MrBayes v 3.2L. depurator COI sequences was reconstructed using the median joining methodThe phylogenetic relationships between http://www.socib.eu/) and buoys located within the different sampling areas  salinity data as a proxy for identifying Atlantic and Mediterranean water masses .


Classical methods for determining malignant disease prognosis are based upon characterizing the morphology and location of tumor cells. While this analysis provides important information about a patient's disease it fails to capture the biological complexity of the tumor microenvironment and the contribution of the anti-tumor immune response. Basic histological quantification of T lymphocyte density, cytotoxicity, and memory by CD3, CD8, and CD45RO, respectively, has demonstrated that increased infiltration of T lymphocytes is associated with statistically significant improvement in patients' disease-free survival (DFS) and overall survival (OS) in colorectal carcinoma (CRC). Delineating the location of T lymphocytes into two areas within the primary tumor, the center and the invading margin is termed Immunoscore.In this study we examined whether tumor genotype could predict the clinical outcome of patients with CRC and if combining Immunoscore with analysis of the tumor genotype could further enhance the accuracy of predicting clinical outcome. We analyzed 200 primary colorectal tumor samples for mutations in 50 common driver genes, including those implicated in models of colon carcinogenesis . Each mutation was then assessed for prediction of DFS. Patients presented with a mean of 4 ± 2 mutations. The most common mutations were in KRAS (58%), TP53 (49%) and APC (37%). Neither, a single mutation or the number of mutations significantly predicted clinical outcome.We examined whether mutations in the primary tumor were associated with presence of metastasis. Mutational profiles were remarkably similar between primary tumors from patients whether or not they had metastatic progression at the time of surgery. None of the 50 major cancer genes was more frequently mutated in patients with metastasis. However, using integrative analysis we found that tumors from patients with metastatic disease downregulated adaptive immune genes including those involved in co-stimulation, proliferation and positive regulation of T cells. Furthermore when analyzing the ability to predict patients' OS using Immunoscore in these late stage tumors, high Immunoscore still distinguished patients with better clinical prognosis. These data suggest immune contexture accurately predicts clinical outcome regardless of tumor stage. Further analysis of pre-existing immunity together with the analysis of tumor genotype may provide better understanding of tumor progression and invasion.


Nigeria is expected to be the nation with the 11th highest population of older persons by 2015. This demographic transition will have implications for the medical care services. Currently, there is no geriatric social health insurance policy nor comprehensive geriatric healthcare services in the country. The minimal health system response may be due to lack of comprehensive knowledge of geriatric disease burden, pattern and healthcare needs. This paper aims to describe the trend and pattern of geriatric health research publications, and to review the current geriatric health policies in Nigeria.A systematic review of published reports in PubMed was performed for the period of January 1990 to April 2014. The review of current geriatric health policies was conducted by document analyses of an annotated national bibliography on digitized health policies and guidelines.A total of 38 policies and guidelines of the Federal Ministry of Health were reviewed and none of them targeted geriatric health. The systematic review identified 52 eligible studies out of 3519 studies. Twenty-five studies (48%) were community-based studies, twenty (38%) were conducted in hospital settings, while only one was carried out in an old people’s home and four were narrative literature reviews. Twelve studies (23%) were done in a rural area, 24 (46%) in an urban area and 11 (21%) report findings from both locations.One study reported the health of the elderly and economic policies (1), elderly destitution (1), health implication of ageing (1), attitude to ageing (2), care for the elderly (3), quality of life and life satisfaction (3), nutrition (2) and physical activity (1). The morbidity pattern reported from the reviewed articles were as follows: medical morbidities (8), geriatric emergencies and admissions (2), dental problems (5), surgical morbidities (1), nosocomial infection (1), mental health morbidities (6), orthopaedic (2), otorhinolaryngology (3), urology (1) and visual morbidities (5).The findings underscore a geriatric health research and policy needs. It is recommended that resources be invested into collaborative research for the development and implementation of evidence based geriatric health policies.


Cervical spine arthritis is a well-recognized complication of juvenile idiopathic arthritis (JIA). It is usually present in patients with more severe disease.To evaluate the prevalence of symptomatic cervical spine involvement and their association with MRI findings in children with JIA.The study was a retrospective study of all patients with JIA followed at University Children's Hospital Ljubljana from September 2010 to January 2013. We reviewed the charts of 206 patients with JIA diagnosed according to the ILAR criteria. Clinical, laboratory data and MRI of cervical spine where available were collected and reviewed.206 patients, 143 girls (69%) and 63 boys (31%) with JIA were followed at University Children's Hospital Ljubljana from September 2010 to January 2013. Twenty-two out of 206 (11%) patients had pain in the neck and/or limited range of motion of cervical spine. Twelve out of 206 (6%) patients had persistent cervical spine symptoms, MRI of the cervical spine with application of paramagnetic contrast was performed in those. The results revealed inflammation of atlantoaxial (AA) joint without subluxation in 6/206 (3%) patients. Among 6 patients with AA joint inflammation were 5 girls and one boy. Three of them had RF negative polyarthritis, two persistent oligoarthritis and one girl had juvenile psoriatic arthritis. Five out of six (83%) patients were ANA positive and none of them had positive HLA B-27 antigen. In four out of six patients (66%) with AA joint inflammation, the neck pain was an initial manifestation of JIA. Five out of six patients (83%) were treated with anti-TNF alpha agents and demonstrated good clinical recovery.The prevalence of AA joint inflammation in our cohort of patients with JIA was 3%. Cervical spine involvement could present as an initial manifestation of JIA and in 83% of our cases demonstrated good clinical response on treatment with anti-TNF alpha agents.None declared.


Borrelia burgdorferi sensu lato. The most important for human pathogenicity are B.garinii, B.afzelii and B.burgdorferi sensu stricto. Indirect laboratory diagnostics of LB is based on serological methods such as Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western Blot Assay (WB) that detect specific antibodies. Another method in the diagnostics of LB is a new test LymeSpot. This test is based on Enzyme-Linked ImmunoSpot Assay (ELISPOT). We present our first experience with the test LymeSpot.Lyme borreliosis (LB) is multisystem infectious disease with various clinical manifestations. In Europe, LB is caused by gram-negative spirochetal bacteria of the complex B. burgdorferi sl.From November 2013 to June 2014 we performed LymeSpot on the group of 42 patients who were tested for suspicion of LB presence. LymeSpot is based on specific cell immune response of T-lymphocytes after stimulation by Borrelia antigens. The result is a number of antigen specific effector T-cells producing IFN-γ in the form of spots. We compared the results of LymeSpot test with the level of specific IgM and IgG antibodies detected by ELISA and WB with using recombinant antigens From the number of 42 patients we detected positive LymeSpot test only in 4 of them , a cutoff value in 3 patients . From the group of 42 patients were simultaneously performed ELISA and WB in 26 persons. In this group we found positive LymeSpot in 4 patients and cutoff value in 2 patients. In the group of 4 patients with positive LymeSpot was detected higher level of IgM antibodies in 3 patients and only IgG antibodies in 1 patient. They were patients with clinical manifestations of LB who were treated with antibiotics afterwards. Among 20 patients with negative results of LymeSpot these findings correlated with antibody response in 8 patients (40%). In this group we detected seronegative finding (IgM-IgG-) or finding of passing through LB (IgM-IgG+). In other 12 patients with negative LymeSpot test we detected serological finding of beginning (IgM+IgG-) or running LB (IgM+IgG+). They were mostly patients without clear symptoms of LB, patients with long-term positivity of IgM antibodies without clinical manifestations of LB or patients after antibiotic therapy.Our first experience shows that results of LymeSpot test better correlate with clinical findings and LB activity as well as can be effective marker of success of antibiotic therapy.


The majority of distal radius fractures are associated with an ulnar styloid fracture -3. ThereBecause of its close association with the Triangular Fibrocartilage Complex (TFCC), fractures of the ulnar styloid might indicate possible TFCC disruption with subsequent DRUJ instability ,8,9. StuBone fragments resulting from non-union could also provoke irritation and pain ,10 due tAs the results of treatment of chronic instability of DRUJ following a distal radius fracture are more unpredictable , it is a


Breast cancer is the second most lethal cancer in women. Further, death rates for African American women are the highest for any racial/ethnic group. Hormone receptor status is one of the major prognostic factors and a determinant of treatment options for breast cancer, thus suggesting the importance of molecular level characterization for precision treatments. In this study, we have identified transcriptome level differences correlating to receptor specific molecular subtypes of breast cancer in African American women.http://cancergenome.nih.gov/), yielding transcriptome level analysis between four specific subtypes of breast cancer (Table Clinical and gene expression data from 18 African American women samples were obtained from The Cancer Genome Atlas (TCGA: The samples were analyzed using One-way ANOVA with Welch's correction for unequal sample sizes with type 3 sum of squares. Genes with ANOVA p-value < 0.01 and with relative expression fold change > |2.0| were considered significantly altered; this yielded 90 differentially expressed genes between cancer subtypes in our dataset (Figure BRCA1, SMAD3, SMAD4, EGFR and MDC1 genes[MDC1 showed altered expression for all subtypes of breast cancer and a significant p-value for the Luminal A subtype compared to the Triple negative subtype. The MDC1 protein has been previously implicated in the DNA damage response[The biological interaction network included important DNA repair sub-networks consisting of  response and signIn conclusion, our results highlight a significant difference in the transcriptome levels of critical DNA repair proteins among the different breast cancer subtypes in African American women. The differentials that were observed stress the importance of molecular level characterizations to understand this disease. Understanding the protein interactions involved in this network will have a major role in predicting best courses of action and aid in precision medicine-based approaches to treating breast cancer.


We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci , monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve. Parthenogenesis is the process by which an oocyte is stimulated to divide and develop into an embryo without being fertilized. It can be artificially induced by triggering unfertilized eggs to resume meiosis without undergoing cell division, or achieved spontaneously following failed fertilization. The resulting embryos, which contain only maternal chromosomes, are unviable due to poor development of extra embryonic tissues –7. Yet, This study was approved by the Ethics Committee at Shaare Zedek Medical Center (IRB 87/07) for the derivation of HESC lines from genetically affected PGD derived embryos, in compliance with protocols approved by the National Ethics committee.The SMA HESC line was established from a PGD derived embryo that developed into a fully grown hatched blastocyst by day 7 following fertilization. At this stage the inner cell mass (ICM) was mechanically isolated by manual cutting with an ultra-sharp splitting blade (Bioniche). The intact ICM clump was placed on a feeder cell layer of mitomycin C-inactivated mouse embryonic fibroblasts and cultured in HESC media . Outgrowths of proliferating cells were manually propagated using the cut-and-paste method. Following 5–7 passages, the newly established HESC line was further propagated by collagenase type IV (Gibco) and then frozen for future use as described in ..31]. In S1 FigDifferential expression of SNRPN  but not H19  in SZ-SMA5, is different from wild-type HESC control (WT) and corresponds to the maternal only origin of SZ-SMA5 cell line.(TIF)Click here for additional data file.S2 FigSequencing results (shaded) of 4 randomly selected probes from the SNP array  were used to validate the CytoScan 750K array data obtained for SZ-SMA5. For each probe heterozygosity rate , allele variations, and SNP array result are indicated, illustrating high accuracy of the assay for whole genome homozygosity detection.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Several classes of antimicrobial nanoparticles (NPs) and nanosized carriers for antibiotics delivery have proved their effectiveness as alternative agents to combat the antimicrobial resistance experienced with conventional drugs. Gold nanostructures find extensive applications in nano electronics and nanomedicine.The present study involved the synthesis of gold nanoparticles using a sodium cholate, as both reducing and capping agent and characterization by UV- Visible spectroscopy and High ResolutionTransmission Electron Microscopy (HRTEM). The antibacterial activity on significant bacterial species was evaluated by micro broth dilution method. The mechanism for antibacterial action of the gold nanoparticles were studied by reactive oxygen species (ROS) generation that causes oxidative stress to microbial cells and release of intracellular enzyme lactate dehydrogenase into extracellular medium (LDH assay) indicative of loss of cell membrane integrity.80) of the nanoparticles on E. coli, S. typhi, P. aeruginosa, K. pneumoniae, ranged from 20 to 40µg/mL. The ROS generation was directly dependent on the concentration of the nanoparticles and no detectable enzyme leakage was recorded.The synthesized gold nanoparticles showed characteristic peak at 541 nm by UV- Visible spectroscopy with particle size of 10nm as confirmed by HRTEM. The minimum inhibitory concentration (MICThe results showed appreciable antibacterial activity of gold nanoparticles against the tested species and the possible mechanism of antibacterial activity may be due to increased intracellular ROS generation causing oxidative stress to the bacterial cells while LDH assay indicated that nanoparticles caused no damage to the cell membrane integrity.


Within the focal point of focused ultrasound (FUS), high pressure fluctuations cause shear stress on cells and tissues with energy dissipation leading to heating of the targeted tissue. The extent of the mechanical and thermal effects strongly depends on the ultrasound parameters and also potentially the acoustic properties of the tumor. High temperatures lead to coagulative necrosis with little immunogenic response as tumor antigens most likely denature during the ablation. As antigen presentation by activated dendritic cells (DCs) is necessary to activate T cells during an anti-tumor immune response, we hypothesize that non-ablative FUS treatment can be used to increase tumor immunogenicity by activating molecules that enhance intratumor dendritic cell (DC) infiltration and T cell activation. One of the first steps in this process is an activated stress response, including surface calreticulin (CRT), high mobility group protein B1 (HMGB1) release, ATP secretion and heat shock protein 70 (HSP70) surface expression and release. Using non-ablative FUS, antigen release can be stimulated by mechanical stress, thermal stress, or both. Here, we examine the stress response following non-ablative FUS treatment in three murine tumor models with varying consistencies and determine the causative agent  of the stress.in vivo in three subcutaneous tumor models in C57BL/6 mice, varying the mechanical and thermal energies to determine which protocol results in the greatest anti-tumor immunity. We investigated CRT surface expression, ATP release and both intra and extracellular expression/release of HMGB1 and HSP70. Since the effect of FUS treatment may vary depending on the cancer cell type, we investigated three different mouse cancer cell lines varying in consistency from solid to fluid . Animals were sacrificed 24 hours post-treatment and tumors were excised for stress response analysis by flow cytometry. Plasma was also obtained for analysis of soluble proteins by ELISA. Stress response was compared to non-treated tumors (control) as well as to in vitro treatment of a cell pellet with the same FUS parameters. For all FUS treatment a Philips Therapy and Imaging Probe System  was used.We tested a range of non-ablative FUS treatment protocols in vivo model of 3LL 24 hours following FUS treatment that delivers high thermal energy when compared to other treatment settings and no treatment  in surface expression of HSP70 in an