Short pyrosequencing reads suffice for accurate microbial community analysis

Pyrosequencing technology allows us to characterize microbial communities using 16S ribosomal RNA (rRNA) sequences orders of magnitude faster and more cheaply than has previously been possible. However, results from different studies using pyrosequencing and traditional sequencing are often difficult to compare, because amplicons covering different regions of the rRNA might yield different conclusions. We used sequences from over 200 globally dispersed environments to test whether studies that used similar primers clustered together mistakenly, without regard to environment. We then tested whether primer choice affects sequence-based community analyses using UniFrac, our recently-developed method for comparing microbial communities. We performed three tests of primer effects. We tested whether different simulated amplicons generated the same UniFrac clustering results as near-full-length sequences for three recent large-scale studies of microbial communities in the mouse and human gut, and the Guerrero Negro microbial mat. We then repeated this analysis for short sequences (100-, 150-, 200- and 250-base reads) resembling those produced by pyrosequencing. The results show that sequencing effort is best focused on gathering more short sequences rather than fewer longer ones, provided that the primers are chosen wisely, and that community comparison methods such as UniFrac are surprisingly robust to variation in the region sequenced

MixHMM: Inferring Copy Number Variation and Allelic Imbalance Using SNP Arrays and Tumor Samples Mixed with Stromal Cells

BACKGROUND: Genotyping platforms such as single nucleotide polymorphism (SNP) arrays are powerful tools to study genomic aberrations in cancer samples. Allele specific information from SNP arrays provides valuable information for interpreting copy number variation (CNV) and allelic imbalance including loss-of-heterozygosity (LOH) beyond that obtained from the total DNA signal available from array comparative genomic hybridization (aCGH) platforms. Several algorithms based on hidden Markov models (HMMs) have been designed to detect copy number changes and copy-neutral LOH making use of the allele information on SNP arrays. However heterogeneity in clinical samples, due to stromal contamination and somatic alterations, complicates analysis and interpretation of these data. METHODS: We have developed MixHMM, a novel hidden Markov model using hidden states based on chromosomal structural aberrations. MixHMM allows CNV detection for copy numbers up to 7 and allows more complete and accurate description of other forms of allelic imbalance, such as increased copy number LOH or imbalanced amplifications. MixHMM also incorporates a novel sample mixing model that allows detection of tumor CNV events in heterogeneous tumor samples, where cancer cells are mixed with a proportion of stromal cells. CONCLUSIONS: We validate MixHMM and demonstrate its advantages with simulated samples, clinical tumor samples and a dilution series of mixed samples. We have shown that the CNVs of cancer cells in a tumor sample contaminated with up to 80% of stromal cells can be detected accurately using Illumina BeadChip and MixHMM. AVAILABILITY: The MixHMM is available as a Python package provided with some other useful tools at http://genecube.med.yale.edu:8080/MixHMM

In vitro study on human cytomegalovirus affecting early pregnancy villous EVT's invasion function

<p>Abstract</p> <p>Background</p> <p>Human cytomegalovirus (HCMV) is the most common pathogen in uterus during pregnancy, which may lead to some serious results such as miscarriage, stillbirth, cerebellar malformation, fetus developmental retardation, but its pathogenesis has not been fully explained. The hypofunction of extravillous cytotrophoblast (EVT) invasion is the essential pathologic base of some complications of pregnancy. c-erbB-2 is a kind of oncogene protein and closely linked with embryogenesis, tissue repair and regeneration. Matrix metalloproteinase (MMP) is one of the key enzymes which affect EVT migration and invasion function. The expression level changes of c-erbB-2, MMP-2 and MMP-9 can reflect the changes of EVT invasion function.</p> <p>Results</p> <p>To explore the influence of HCMV on the invasion function of EVT, we tested the protein expression level changes of c-erbB-2, MMP-2 and MMP-9 in villous explant cultured in vitro infected by HCMV, with the use of immunohistochemistry SP method and western blot. We confirmed that HCMV can reproduce and spread in early pregnancy villus; c-erbB-2 protein mainly expressed in normal early pregnancy villous syncytiotrophoblast (ST) remote plasma membrane and EVT, especially remote EVT cell membrane in villous stem cell column, little expressed in ST proximal end cell membrane and interstitial cells; MMP-2 protein primarily expressed in early pregnancy villous EVT endochylema and rarely in villous trophoblast (VT), ST and interstitial cells; MMP-9 protein largely expressed in early pregnancy villous mesenchyme, EVT and VT endochylema. Compared with control group, the three kinds of protein expression level in early pregnancy villus of virus group significantly decreased (P < 0.05).</p> <p>Conclusion</p> <p>HCMV can infect villus in vitro and cause the decrease of early pregnancy villous EVT's invasion function.</p

PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth.

p53 acetylation is indispensable for its transcriptional activity and tumor suppressive function. However, the identity of reader protein(s) for p53 acetylation remains elusive. PBRM1, the second most highly mutated tumor suppressor gene in kidney cancer, encodes PBRM1. Here, we identify PBRM1 as a reader for p53 acetylation on lysine 382 (K382Ac) through its bromodomain 4 (BD4). Notably, mutations on key residues of BD4 disrupt recognition of p53 K382Ac. The mutation in BD4 also reduces p53 binding to promoters of target genes such as CDKN1A (p21). Consequently, the PBRM1 BD4 mutant fails to fully support p53 transcriptional activity and is defective as a tumor suppressor. We also find that expressions of PBRM1 and p21 correlate with each other in human kidney cancer samples. Our findings uncover a tumor suppressive mechanism of PBRM1 in kidney cancer and provide a mechanistic insight into the crosstalk between p53 and SWI/SNF complexes

Murine model for congenital CMV infection and hearing impairment

<p>Abstract</p> <p>Background</p> <p>Congenital cytomegalovirus (CMV) infection is the leading cause of sensorineural hearing loss (SNHL), and SNHL is the most frequent sequela of congenital CMV infection. But the pathogenic mechanism remains unknown, and there is no ideal CMV intrauterine infection animal model to study the mechanisms by which SNHL develops.</p> <p>Methods</p> <p>We established the congenital murine cytomegalovirus (MCMV) infection model by directly injecting the virus into the placenta on day 12.5 of gestation. Then, we observed the development and the MCMV congenital infection rate of the fetuses on the day they were born. Furthermore, we detected the auditory functions, the conditions of the MCMV infection, and the histological change of the inner ears of 28-day-old and 70-day-old offspring.</p> <p>Results</p> <p>Both the fetal loss rate and the teratism rate of offspring whose placentas were inoculated with MCMV increased, and their body length, head circumference, and weight decreased. The hearing level of offspring both decreased at both 28- and 70-days post birth; the 70-day-old mice developed lower hearing levels than did the 28-day old mice. No significant inflammatory changes in the cochleae of the mice were observed. MCMV DNA signals were mainly detected in the spiral ganglion neurons and the endolymph area, but not in the perilymph area. The number of neurons decreased, and their ultrastructures changed. Moreover, with age, the number of neurons dramatically decreased, and the ultrastructural lesions of neurons became much more severe.</p> <p>Conclusions</p> <p>The results suggest that the direct injection of MCMV into the placenta may efficiently cause fetal infection and disturb the intrauterine development of the fetus, and placental inoculation itself has no obvious adverse effects on offspring. The reduction in the number of spiral ganglion neurons and the ultrastructural lesions of the neurons may be the major cause of congenital CMV infection-induced progressive SNHL.</p

Incorporating Traffic Control and Safety Hardware Performance Functions into Risk-based Highway Safety Analysis

Traffic control and safety hardware such as traffic signs, lighting, signals, pavement markings, guardrails, barriers, and crash cushions form an important and inseparable part of highway infrastructure affecting safety performance. Significant progress has been made in recent decades to develop safety performance functions and crash modification factors for site-specific crash predictions. However, the existing models and methods lack rigorous treatments of safety impacts of time-deteriorating conditions of traffic control and safety hardware. This study introduces a refined method for computing the Safety Index (SI) as a means of crash predictions for a highway segment that incorporates traffic control and safety hardware performance functions into the analysis. The proposed method is applied in a computation experiment using five-year data on nearly two hundred rural and urban highway segments. The root-mean square error (RMSE), Chi-square, Spearman’s rank correlation, and Mann-Whitney U tests are employed for validation.</p

Spontaneous tumour regression in keratoacanthomas is driven by Wnt/retinoic acid signalling cross-talk

A fundamental goal in cancer biology is to identify the cells and signalling pathways that are keys to induce tumour regression. Here we use a spontaneously self-regressing tumour, cutaneous keratoacanthoma (KAs), to identify physiological mechanisms that drive tumour regression. By using a mouse model system that recapitulates the behaviour of human KAs, we show that self-regressing tumours shift their balance to a differentiation programme during regression. Furthermore, we demonstrate that developmental programs utilized for skin hair follicle regeneration, such as Wnt, are hijacked to sustain tumour growth and that the retinoic acid (RA) signalling pathway promotes tumour regression by inhibiting Wnt signalling. Finally, we find that RA signalling can induce regression of malignant tumours that do not normally spontaneously regress, such as squamous cell carcinomas. These findings provide new insights into the physiological mechanisms of tumour regression and suggest therapeutic strategies to induce tumour regression. © 2014 Macmillan Publishers Limited

Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer.

Whereas VHL inactivation is a primary event in clear cell renal cell carcinoma (ccRCC), the precise mechanism(s) of how this interacts with the secondary mutations in tumor suppressor genes, including PBRM1, KDM5C/JARID1C, SETD2, and/or BAP1, remains unclear. Gene expression analyses reveal that VHL, PBRM1, or KDM5C share a common regulation of interferon response expression signature. Loss of HIF2α, PBRM1, or KDM5C in VHL-/-cells reduces the expression of interferon stimulated gene factor 3 (ISGF3), a transcription factor that regulates the interferon signature. Moreover, loss of SETD2 or BAP1 also reduces the ISGF3 level. Finally, ISGF3 is strongly tumor-suppressive in a xenograft model as its loss significantly enhances tumor growth. Conversely, reactivation of ISGF3 retards tumor growth by PBRM1-deficient ccRCC cells. Thus after VHL inactivation, HIF induces ISGF3, which is reversed by the loss of secondary tumor suppressors, suggesting that this is a key negative feedback loop in ccRCC. © 2018, Liao et al