Biology subjects

Shah, P.

Publications and source records attributed to Shah, P..

101 records · Page 6Linked to original sources

bioRxiv2018

Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

RNA binding proteins, such as IMP1, are emerging as essential regulators of intestinal development and cancer. IMP1 hypomorphic mice exhibit severe intestinal growth defects, yet its role in adult intestinal epithelium is unclear. We employed ribosome profiling to test the effect of IMP1 loss on the \"translatome\" in colon cancer cell lines. In parallel, we evaluated mice with intestinal epithelial-specific Imp1 deletion (Imp1{Delta}IEC) following irradiation or colitis models. Ribosome-profiling revealed translation efficiency changes for multiple pathways important for intestinal homeostasis, including autophagy, in IMP1 knockout cells. We found increased autophagy flux in Imp1{Delta}IEC mice, reinforced through in silico and biochemical analyses revealing direct binding of IMP1 to autophagy transcripts MAP1LC3B and ATG3. We found that Imp1{Delta}IEC mice exhibit enhanced recovery following irradiation, which is attenuated with genetic deletion of autophagy gene Atg7. Finally, we demonstrated that IMP1 is upregulated in Crohns disease patients and Imp1 loss lessened colitis severity in mice. These studies demonstrate that IMP1 acts as a posttranscriptional regulator of gut epithelial repair post-irradiation and colitis, in part through modulation of autophagy.

physiology↗

bioRxiv2018

The ATPase activity of E. coli RecA prevents accumulation of toxic complexes formed by erroneous binding to undamaged double strand DNA

The E. coli RecA protein catalyzes the central step of homologous recombination using its homology search and strand exchange activity. RecA is a DNA-dependent ATPase, but its homology search and strand exchange activities are independent of its ATPase activity. ATP hydrolysis converts a high affinity DNA binding form, RecA-ATP, to a low affinity form RecA-ADP, thereby supporting an ATP hydrolysis-dependent dynamic cycle of DNA binding and dissociation. We provide evidence for a novel function of RecAs dynamic behavior; RecAs ATPase activity prevents accumulation of toxic complexes caused by direct binding of RecA to undamaged regions of dsDNA. We show that a mutant form of RecA, RecA-K250N, previously shown to be toxic to E. coli, is a loss-of-function ATPase-defective mutant. We use a new method for detecting RecA complexes involving nucleoid surface spreading and immunostaining. The method allows detection of damage-induced RecA foci; STED microscopy revealed these to typically be between 50 and 200 nm in length. RecA-K250N, and other toxic variants of RecA, form spontaneous DNA-bound complexes that are independent of replication and of accessory proteins required to load RecA onto tracts of ssDNA in vivo, supporting the hypothesis that RecAs expenditure of ATP serves an error correction function.

genetics↗

bioRxiv2018

MS-PyCloud: An open-source, cloud computing-based pipeline for LC-MS/MS data analysis

Rapid development and wide adoption of mass spectrometry-based proteomics technologies have empowered scientists to study proteins and their modifications in complex samples on a large scale. This progress has also created unprecedented challenges for individual labs to store, manage and analyze proteomics data, both in the cost for proprietary software and high-performance computing, and the long processing time that discourages on-the-fly changes of data processing settings required in explorative and discovery analysis. We developed an open-source, cloud computing-based pipeline, MS-PyCloud, with graphical user interface (GUI) support, for LC-MS/MS data analysis. The major components of this pipeline include data file integrity validation, MS/MS database search for spectral assignment, false discovery rate estimation, protein inference, determination of protein post-translation modifications, and quantitation of specific (modified) peptides and proteins. To ensure the transparency and reproducibility of data analysis, MS-PyCloud includes open source software tools with comprehensive testing and versioning for spectrum assignments. Leveraging public cloud computing infrastructure via Amazon Web Services (AWS), MS-PyCloud scales seamlessly based on analysis demand to achieve fast and efficient performance. Application of the pipeline to the analysis of large-scale iTRAQ/TMT LC-MS/MS data sets demonstrated the effectiveness and high performance of MS-PyCloud. The software can be downloaded at: https://bitbucket.org/mschnau/ms-pycloud/downloads/

bioinformatics↗

bioRxiv2018

The cellular NMD pathway restricts Zika virus infection and is targeted by the viral capsid protein

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly1, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice2-4. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1)5. Endogenous UPF1 interacted with the viral capsid protein in co-immunoprecipitation experiments and capsid expression post-transcriptionally downregulated UPF1, a process that we confirmed occurs during de novo ZIKV infection. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures. We therefore propose that ZIKV, via the capsid protein, has evolved a strategy to dampen antiviral activities of NMD6,7, which subsequently contributes to neuropathology in vivo.

microbiology↗

bioRxiv2017

Reanalysis of global proteomic and phosphoproteomic data identified a large number of glycopeptides

Protein glycosylation plays fundamental roles in many cellular processes, and previous reports have shown dysregulation to be associated with several human diseases, including diabetes, cancer, and neurodegenerative disorders. Despite the vital role of glycosylation for proper protein function, the analysis of glycoproteins has been lagged behind to other protein modifications. In this study, we describe the re-analysis of global proteomic data from breast cancer xenograft tissues using recently developed software package GPQuest 2.0, revealing a large number of previously unidentified N-linked glycopeptides. More importantly, we found that using immobilized metal affinity chromatography (IMAC) technology for the enrichment of phosphopeptides had co-enriched a substantial number of sialoglycopeptides, allowing for a large-scale analysis of sialoglycopeptides in conjunction with the analysis of phosphopeptides. Collectively, combined MS/MS analyses of global proteomic and phosphoproteomic datasets resulted in the identification of 6,724 N-linked glycopeptides from 617 glycoproteins derived from two breast cancer xenograft tissues. Next, we utilized GPQuest for the re-analysis of global and phosphoproteomic data generated from 108 human breast cancer tissues that were previously analyzed by Clinical Proteomic Analysis Consortium (CPTAC). Reanalysis of the CPTAC dataset resulted in the identification of 2,683 glycopeptides from the global proteomic data set and 4,554 glycopeptides from phosphoproteomic data set, respectively. Together, 11,292 N-linked glycopeptides corresponding to 1,731 N-linked glycosites from 883 human glycoproteins were identified from the two data sets. This analysis revealed an extensive number of glycopeptides hidden in the global and enriched in IMAC-based phosphopeptide-enriched proteomic data, information which would have remained unknown from the original study otherwise. The reanalysis described herein can be readily applied to identify glycopeptides from already existing data sets, providing insight into many important facets of protein glycosylation in different biological, physiological, and pathological processes.

systems biology↗

bioRxiv2017

Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short read whole genome sequencing. However, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only [~]1ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as Linked-Reads. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. 2017). In this manuscript, we show the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN1 and SMN2. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

genomics↗

bioRxiv2017

Reference Quality Assembly of the 3.5 Gb genome of Capsicum annuum from a Single Linked-Read Library

BackgroundLinked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Capsicum annuum) genome with a single Linked-Read library. Plant genomes, including pepper, are characterized by long, highly similar repetitive sequences. Accordingly, significant effort is used to ensure the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus. With a phased assembly approach, we targeted a heterozygous F1 derived from a wide cross to assess the ability to derive both haplotypes for a pungency gene characterized by a large insertion/deletion.\n\nResultsThe Supernova software generated a highly ordered, more contiguous sequence assembly than all currently available C. annuum reference genomes. Eighty-four percent of the final assembly was anchored and oriented using four de novo linkage maps. A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly. The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5 kb insertion/deletion haplotypes of the PUN1 locus in the F1 sample that represents pungent and non-pungent peppers.\n\nConclusionsThe most contiguous pepper genome assembly to date has been generated through this work which demonstrates that Linked-Read library technology provides a rapid tool to assemble de novo complex highly repetitive heterozygous plant genomes. This technology can provide an opportunity to cost-effectively develop high-quality reference genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction.

genomics↗

bioRxiv2017

A simple open-source method for highly multiplexed imaging of single cells in tissues and tumours

The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.

cancer biology↗

bioRxiv2017

Improved de novo Genome Assembly: Synthetic long read sequencing combined with optical mapping produce a high quality mammalian genome at relatively low cost

Current short-read methods have come to dominate genome sequencing because they are cost-effective, rapid, and accurate. However, short reads are most applicable when data can be aligned to a known reference. Two new methods for de novo assembly are linked-reads and restriction-site labeled optical maps. We combined commercial applications of these technologies for genome assembly of an endangered mammal, the Hawaiian Monk seal.\n\nWe show that the linked-reads produced with 10X Genomics Chromium chemistry and assembled with Supernova v1.1 software produced scaffolds with an N50 of 22.23 Mbp with the longest individual scaffold of 84.06 Mbp. When combined with Bionano Genomics optical maps using Bionano RefAligner, the scaffold N50 increased to 29.65 Mbp for a total of 170 hybrid scaffolds, the longest of which was 84.78 Mbp. These results were 161X and 215X, respectively, improved over DISCOVAR de novo assemblies. The quality of the scaffolds was assessed using conserved synteny analysis of both the DNA sequence and predicted seal proteins relative to the genomes of humans and other species. We found large blocks of conserved synteny suggesting that the hybrid scaffolds were high quality. An inversion in one scaffold complementary to human chromosome 6 was found and confirmed by optical maps.\n\nThe complementarity of linked-reads and optical maps is likely to make the production of high quality genomes more routine and economical and, by doing so, significantly improve our understanding of comparative genome biology.

genomics↗

bioRxiv2017

Assessing Inhibitors Of Mutant Isocitrate Dehydrogenase Using A Suite Of Pre-Clinical Discovery Assays

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation and secretion of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). Here, a panel of mIDH inhibitors were systematically profiled using biochemical, cell-based, and tier-one ADME techniques. We quantified the biochemical effect of each inhibitor on mIDH1 (R132H and R132C) and mIDH2 (R172Q). The effect of these inhibitors on 2-HG concentrations in seven cell lines representing five different IDH1 mutations in both 2D and 3D cell cultures was assessed. Target engagement of these inhibitors was analyzed utilizing cellular thermal shift assays (CETSA), the effects of inhibitors on reversing 2-HG-induced block on leukemic cellular differentiation. We conclude from our mIDH1 assay panel that AG-120 and a Novartis inhibitor exhibited excellent activity in all biochemical and most cellular assays. While AG-120 has superior DMPK properties, it lacks efficacy a leukemic differentiation model. In conclusion, we present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors.

cancer biology↗

bioRxiv2017

riboviz: analysis and visualization of ribosome profiling datasets

Using high-throughput sequencing to monitor translation in vivo, ribosome profiling can provide critical insights into the dynamics and regulation of protein synthesis in a cell. Since its introduction in 2009, this technique has played a key role in driving biological discovery, and yet it requires a rigorous computational toolkit for widespread adoption. We developed a processing pipeline and browser-based visualization, riboviz, that allows convenient exploration and analysis of riboseq datasets. In implementation, riboviz consists of a comprehensive and flexible backend analysis pipeline that allows the user to analyze their private unpublished dataset, along with a web application for comparison with previously published public datasets.\n\nAvailability and implementationJavaScript and R source code and extra documentation are freely available from https://github.com/shahpr/RiboViz, while the web-application is live at www.riboviz.org.

bioinformatics↗