TGF-beta 1 induces human alveolar epithelial to mesenchymal cell transition (EMT)

Background: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT. Methods: A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA. Results: The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes. Conclusion: Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon

Brief report: a comparison of the preference for viewing social and non-social movies in typical and autistic adolescents

The recently proposed Social Motivation theory (Chevallier et al., Trends in cognitive sciences 16(4):231–239, 2012) suggests that social difficulties in Autism Spectrum Condition (ASC) might be caused by a difference in the motivation to engage with other people. Here we compared adolescents with (N = 31) and without (N = 37) ASC on the Choose-a-Movie paradigm that measures the social seeking. The results showed a preference for viewing objects over smiling faces in ASC, which is in line with the theory of low social motivation. However, typical adolescents did not show any stimuli preferences, raising questions about developmental changes in social motivation. Age was found to play a significant role in moderating the choice behaviour of the participants. We discuss the implications of these findings in detail

Multifocal VEP (mfVEP) reveals abnormal neuronal delays in diabetes

This pilot study examined the diagnostic role of multifocal visually evoked potentials (mfVEP) in a small number of patients with diabetes. mfVEP, mfERG, and fundus photographs of both eyes of five patients with diabetes, three with nonproliferative diabetic retinopathy (NPDR) and two without NPDR were examined. Thirteen control subjects were also examined. Eighteen zones were constructed from the 60-element mfVEP stimulus array. mfVEP implicit time (IT) and amplitude (SNR) differences were tested between subject groups. We also examined whether there was a difference in function for patches with and without retinopathy in the NPDR group. Lastly, we compared mfVEP and mfERG results in the same patients. We found significant mfVEP IT differences between controls and all patients with diabetes, controls and diabetics without retinopathy, and between controls and diabetics with retinopathy. The subject groups did not differ significantly in terms of SNR. In the retinopathy group, ITs from zones with retinopathy were significantly longer than ITs from zones without retinopathy (P = 0.016). mfERG IT was more frequently abnormal than mfVEP IT. In addition, mfERG hexagons were twice as likely to be abnormal if the corresponding mfVEP zone was abnormal (P < 0.05). mfVEP implicit times are significantly delayed in patients with diabetes even when there is no retinopathy. These cortical response results are similar, albeit considerably less abnormal, than those previously reported for retinal (mfERG) responses in patients with diabetes. A correlation exists between the location of abnormal mfERG hexagons and abnormal mfVEP zones

Identification of errors introduced during high throughput sequencing of the T cell receptor repertoire

<p>Abstract</p> <p>Background</p> <p>Recent advances in massively parallel sequencing have increased the depth at which T cell receptor (TCR) repertoires can be probed by >3log10, allowing for saturation sequencing of immune repertoires. The resolution of this sequencing is dependent on its accuracy, and direct assessments of the errors formed during high throughput repertoire analyses are limited.</p> <p>Results</p> <p>We analyzed 3 monoclonal TCR from TCR transgenic, Rag^-/-mice using Illumina^®sequencing. A total of 27 sequencing reactions were performed for each TCR using a trifurcating design in which samples were divided into 3 at significant processing junctures. More than 20 million complementarity determining region (CDR) 3 sequences were analyzed. Filtering for lower quality sequences diminished but did not eliminate sequence errors, which occurred within 1-6% of sequences. Erroneous sequences were pre-dominantly of correct length and contained single nucleotide substitutions. Rates of specific substitutions varied dramatically in a position-dependent manner. Four substitutions, all purine-pyrimidine transversions, predominated. Solid phase amplification and sequencing rather than liquid sample amplification and preparation appeared to be the primary sources of error. Analysis of polyclonal repertoires demonstrated the impact of error accumulation on data parameters.</p> <p>Conclusions</p> <p>Caution is needed in interpreting repertoire data due to potential contamination with mis-sequence reads. However, a high association of errors with phred score, high relatedness of erroneous sequences with the parental sequence, dominance of specific nt substitutions, and skewed ratio of forward to reverse reads among erroneous sequences indicate approaches to filter erroneous sequences from repertoire data sets.</p

Evolution of Burkholderia pseudomallei in Recurrent Melioidosis

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis

Big Genomes Facilitate the Comparative Identification of Regulatory Elements

The identification of regulatory sequences in animal genomes remains a significant challenge. Comparative genomic methods that use patterns of evolutionary conservation to identify non-coding sequences with regulatory function have yielded many new vertebrate enhancers. However, these methods have not contributed significantly to the identification of regulatory sequences in sequenced invertebrate taxa. We demonstrate here that this differential success, which is often attributed to fundamental differences in the nature of vertebrate and invertebrate regulatory sequences, is instead primarily a product of the relatively small size of sequenced invertebrate genomes. We sequenced and compared loci involved in early embryonic patterning from four species of true fruit flies (family Tephritidae) that have genomes four to six times larger than those of Drosophila melanogaster. Unlike in Drosophila, where virtually all non-coding DNA is highly conserved, blocks of conserved non-coding sequence in tephritids are flanked by large stretches of poorly conserved sequence, similar to what is observed in vertebrate genomes. We tested the activities of nine conserved non-coding sequences flanking the even-skipped gene of the teprhitid Ceratis capitata in transgenic D. melanogaster embryos, six of which drove patterns that recapitulate those of known D. melanogaster enhancers. In contrast, none of the three non-conserved tephritid non-coding sequences that we tested drove expression in D. melanogaster embryos. Based on the landscape of non-coding conservation in tephritids, and our initial success in using conservation in tephritids to identify D. melanogaster regulatory sequences, we suggest that comparison of tephritid genomes may provide a systematic means to annotate the non-coding portion of the D. melanogaster genome. We also propose that large genomes be given more consideration in the selection of species for comparative genomics projects, to provide increased power to detect functional non-coding DNAs and to provide a less biased view of the evolution and function of animal genomes

Progesterone Inhibits Epithelial-to-Mesenchymal Transition in Endometrial Cancer

Background: Every year approximately 74,000 women die of endometrial cancer, mainly due to recurrent or metastatic disease. The presence of tumor infiltrating lymphocytes (TILs) as well as progesterone receptor (PR) positivity has been correlated with improved prognosis. This study describes two mechanisms by which

Identification and characterisation of novel SNP markers in Atlantic cod: Evidence for directional selection

<p>Abstract</p> <p>Background</p> <p>The Atlantic cod (<it>Gadus morhua</it>) is a groundfish of great economic value in fisheries and an emerging species in aquaculture. Genetic markers are needed to identify wild stocks in order to ensure sustainable management, and for marker-assisted selection and pedigree determination in aquaculture. Here, we report on the development and evaluation of a large number of Single Nucleotide Polymorphism (SNP) markers from the alignment of Expressed Sequence Tag (EST) sequences in Atlantic cod. We also present basic population parameters of the SNPs in samples of North-East Arctic cod and Norwegian coastal cod obtained from three different localities, and test for SNPs that may have been targeted by natural selection.</p> <p>Results</p> <p>A total of 17,056 EST sequences were used to find 724 putative SNPs, from which 318 segregating SNPs were isolated. The SNPs were tested on Atlantic cod from four different sites, comprising both North-East Arctic cod (NEAC) and Norwegian coastal cod (NCC). The average heterozygosity of the SNPs was 0.25 and the average minor allele frequency was 0.18. <it>F</it>_{<it>ST </it>}values were highly variable, with the majority of SNPs displaying very little differentiation while others had <it>F</it>_{<it>ST </it>}values as high as 0.83. The <it>F</it>_{<it>ST </it>}values of 29 SNPs were found to be larger than expected under a strictly neutral model, suggesting that these loci are, or have been, influenced by natural selection. For the majority of these outlier SNPs, allele frequencies in a northern sample of NCC were intermediate between allele frequencies in a southern sample of NCC and a sample of NEAC, indicating a cline in allele frequencies similar to that found at the Pantophysin I locus.</p> <p>Conclusion</p> <p>The SNP markers presented here are powerful tools for future genetics work related to management and aquaculture. In particular, some SNPs exhibiting high levels of population divergence have potential to significantly enhance studies on the population structure of Atlantic cod.</p

Identifying Causal Genes and Dysregulated Pathways in Complex Diseases

In complex diseases, various combinations of genomic perturbations often lead to the same phenotype. On a molecular level, combinations of genomic perturbations are assumed to dys-regulate the same cellular pathways. Such a pathway-centric perspective is fundamental to understanding the mechanisms of complex diseases and the identification of potential drug targets. In order to provide an integrated perspective on complex disease mechanisms, we developed a novel computational method to simultaneously identify causal genes and dys-regulated pathways. First, we identified a representative set of genes that are differentially expressed in cancer compared to non-tumor control cases. Assuming that disease-associated gene expression changes are caused by genomic alterations, we determined potential paths from such genomic causes to target genes through a network of molecular interactions. Applying our method to sets of genomic alterations and gene expression profiles of 158 Glioblastoma multiforme (GBM) patients we uncovered candidate causal genes and causal paths that are potentially responsible for the altered expression of disease genes. We discovered a set of putative causal genes that potentially play a role in the disease. Combining an expression Quantitative Trait Loci (eQTL) analysis with pathway information, our approach allowed us not only to identify potential causal genes but also to find intermediate nodes and pathways mediating the information flow between causal and target genes. Our results indicate that different genomic perturbations indeed dys-regulate the same functional pathways, supporting a pathway-centric perspective of cancer. While copy number alterations and gene expression data of glioblastoma patients provided opportunities to test our approach, our method can be applied to any disease system where genetic variations play a fundamental causal role

Protein Networks Reveal Detection Bias and Species Consistency When Analysed by Information-Theoretic Methods

We apply our recently developed information-theoretic measures for the characterisation and comparison of protein–protein interaction networks. These measures are used to quantify topological network features via macroscopic statistical properties. Network differences are assessed based on these macroscopic properties as opposed to microscopic overlap, homology information or motif occurrences. We present the results of a large–scale analysis of protein–protein interaction networks. Precise null models are used in our analyses, allowing for reliable interpretation of the results. By quantifying the methodological biases of the experimental data, we can define an information threshold above which networks may be deemed to comprise consistent macroscopic topological properties, despite their small microscopic overlaps. Based on this rationale, data from yeast–two–hybrid methods are sufficiently consistent to allow for intra–species comparisons (between different experiments) and inter–species comparisons, while data from affinity–purification mass–spectrometry methods show large differences even within intra–species comparisons