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Correction: Defining the microbial transcriptional response to colitis through integrated host and microbiome profiling.
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Embryonic progenitor pools generate diversity in fine-scale excitatory cortical subnetworks.
The mammalian neocortex is characterized by a variety of neuronal cell types and precise arrangements of synaptic connections, but the processes that generate this diversity are poorly understood. Here we examine how a pool of embryonic progenitor cells consisting of apical intermediate progenitors (aIPs) contribute to diversity within the upper layers of mouse cortex. In utero labeling combined with single-cell RNA-sequencing reveals that aIPs can generate transcriptionally defined glutamatergic cell types, when compared to neighboring neurons born from other embryonic progenitor pools. Whilst sharing layer-associated morphological and functional properties, simultaneous patch clamp recordings and optogenetic studies reveal that aIP-derived neurons exhibit systematic biases in both their intralaminar monosynaptic connectivity and the post-synaptic partners that they target within deeper layers of cortex. Multiple cortical progenitor pools therefore represent an important factor in establishing diversity amongst local and long-range fine-scale glutamatergic connectivity, which generates subnetworks for routing excitatory synaptic information.
CGAT-core: A python framework for building scalable, reproducible computational biology workflows [version 1; peer review: 1 approved, 1 approved with reservations]
© 2019 Cribbs AP et al. In the genomics era computational biologists regularly need to process, analyse and integrate large and complex biomedical datasets. Analysis inevitably involves multiple dependent steps, resulting in complex pipelines or workflows, often with several branches. Large data volumes mean that processing needs to be quick and efficient and scientific rigour requires that analysis be consistent and fully reproducible. We have developed CGAT-core, a python package for the rapid construction of complex computational workflows. CGAT-core seamlessly handles parallelisation across high performance computing clusters, integration of Conda environments, full parameterisation, database integration and logging. To illustrate our workflow framework, we present a pipeline for the analysis of RNAseq data using pseudo-alignment.
Defining the microbial transcriptional response to colitis through integrated host and microbiome profiling.
The gut microbiome is significantly altered in inflammatory bowel diseases, but the basis of these changes is not well understood. We have combined metagenomic and metatranscriptomic profiling of the gut microbiome to assess modifications to both bacterial community structure and transcriptional activity in a mouse model of colitis. By using transcriptomic analysis of colonic tissue and luminal RNA derived from the host, we have also characterised how host transcription relates to the microbial transcriptional response in inflammation. In colitis, increased abundance and transcription of diverse microbial gene families involved in responses to nutrient deprivation, antimicrobial peptide production and oxidative stress support an adaptation of multiple commensal genera to withstand a diverse set of environmental stressors in the inflammatory environment. These data are supported by a transcriptional signature of activated macrophages and granulocytes in the gut lumen during colitis, a signature that includes the transcription of the key antimicrobial genes S100a8 and S100a9 (calprotectin). Genes involved in microbial resistance to oxidative stress, including Dps/ferritin, Fe-dependent peroxidase and glutathione S-transferase were identified as changing to a greater extent at the level of transcription than would be predicted by DNA abundance changes, implicating a role for increased oxygen tension and/or host-derived reactive oxygen species in driving transcriptional changes in commensal microbes.
Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4.
Signalling through the Hippo (Hpo) pathway involves a kinase cascade, which leads to the phosphorylation and inactivation of the pro-growth transcriptional co-activator Yorkie (Yki). Despite the identification of a large number of pathway members and modulators, our understanding of the molecular events that lead to activation of Hpo and the downstream kinase Warts (Wts) remain incomplete. Recently, targeted degradation of several Hpo pathway components has been demonstrated as a means of regulating pathway activity. In particular, the stability of scaffold protein Salvador (Sav), which is believed to promote Hpo/Wts association, is crucially dependent on its binding partner Hpo. In a cell-based RNAi screen for ubiquitin regulators involved in Sav stability, we identify the HECT domain protein Herc4 (HECT and RLD domain containing E3 ligase) as a Sav E3 ligase. Herc4 expression promotes Sav ubiquitylation and degradation, while Herc4 depletion stabilises Sav. Interestingly, Hpo reduces Sav/Herc4 interaction in a kinase-dependent manner. This suggests the existence of a positive feedback loop, where Hpo stabilises its own positive regulator by antagonising Herc4-mediated degradation of Sav.
Functional viability profiles of breast cancer.
UNLABELLED: The design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging, meaning that alternative strategies such as exploiting synthetic lethality may be beneficial. To address these issues, we have carried out a functional genetic screen in >30 commonly used models of breast cancer to identify genes critical to the growth of specific breast cancer subtypes. In particular, we describe potential new therapeutic targets for PTEN-mutated cancers and for estrogen receptor-positive breast cancers. We also show that large-scale functional profiling allows the classification of breast cancers into subgroups distinct from established subtypes. SIGNIFICANCE: Despite the wealth of molecular profiling data that describe breast tumors and breast tumor cell models, our understanding of the fundamental genetic dependencies in this disease is relatively poor. Using high-throughput RNA interference screening of a series of pharmacologically tractable genes, we have generated comprehensive functional viability profiles for a wide panel of commonly used breast tumor cell models. Analysis of these profiles identifies a series of novel genetic dependencies, including that of PTEN-null breast tumor cells upon mitotic checkpoint kinases, and provides a framework upon which additional dependencies and candidate therapeutic targets may be identified.
De novo point mutations in patients diagnosed with ataxic cerebral palsy.
Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies.
Next-generation sequencing of advanced prostate cancer treated with androgen-deprivation therapy.
BACKGROUND: Androgen-deprivation therapy (ADT) is standard treatment for locally advanced or metastatic prostate cancer (PCa). Many patients develop castration resistance (castration-resistant PCa [CRPC]) after approximately 2-3 yr, with a poor prognosis. The molecular mechanisms underlying CRPC progression are unclear. OBJECTIVE: To undertake quantitative tumour transcriptome profiling prior to and following ADT to identify functionally important androgen-regulated pathways or genes that may be reactivated in CRPC. DESIGN, SETTING, AND PARTICIPANTS: RNA sequencing (RNA-seq) was performed on tumour-rich, targeted prostatic biopsies from seven patients with locally advanced or metastatic PCa before and approximately 22 wk after ADT initiation. Differentially regulated genes were identified in treatment pairs and further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on cell lines and immunohistochemistry on a separate CRPC patient cohort. Functional assays were used to determine the effect of pathway modulation on cell phenotypes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We searched for gene expression changes affecting key cell signalling pathways that may be targeted as proof of principle in a CRPC in vitro cell line model. RESULTS AND LIMITATIONS: We identified ADT-regulated signalling pathways, including the Wnt/β-catenin signalling pathway, and observed overexpression of β-catenin in a subset of CRPC by immunohistochemistry. We validated 6 of 12 (50%) pathway members by qRT-PCR on LNCaP/LNCaP-AI cell RNAs, of which 4 (67%) demonstrated expression changes consistent with RNA-seq data. We show that the tankyrase inhibitor XAV939 (which promotes β-catenin degradation) reduced androgen-independent LNCaP-AI cell line growth compared with androgen-responsive LNCaP cells via an accumulation of cell proportions in the G0/G1 phase and reduction in the S and G2/M phases. Our biopsy protocol did not account for tumour heterogeneity, and pathway inhibition was limited to pharmacologic approaches. CONCLUSIONS: RNA-seq of paired PCa samples revealed ADT-regulated signalling pathways. Proof-of-principle inhibition of the Wnt/β-catenin signalling pathway specifically delays androgen-independent PCa cell cycle progression and proliferation and warrants further investigation as a potential target for therapy for CRPC.
Deep Brain Stimulation in Three Related Cases of North Sea Progressive Myoclonic Epilepsy from South Africa.
We report on a white Afrikaans family from eastern South Africa with three members affected with North Sea progressive myoclonus epilepsy, resulting from a homozygous founder GOSR2 mutation (c.430G>T, p.Gly144Trp). The mutation was identified by exomic sequencing in a research study investigating childhood onset ataxias. All three subjects presented with ataxia, tremor, early gait difficulties, and myoclonic and generalized tonic clonic (GTC) epilepsy. Each patient underwent deep brain stimulation of the caudal Zona Incerta before coming to the attention of the authors. In each case there was a reduction in GTC seizures, and two patients exhibited a reduction in involuntary movements, as evaluated during long-term follow-up. In one case there was an improvement in gait and stance when assessed while the stimulation was on.