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Systems Biology
Addressing Interdisciplinary Difficulties in Developmental Biology/Mathematical Collaborations: A Neural Crest Example.
Apr 12, 2019   Methods In Molecular Biology (Clifton, N.J.)
Newgreen DF, Landman KA, Osborne JM
Addressing Interdisciplinary Difficulties in Developmental Biology/Mathematical Collaborations: A Neural Crest Example.
Apr 12, 2019
Methods In Molecular Biology (Clifton, N.J.)
Mathematical modeling can allow insight into the biological processes that can be difficult to access by conventional biological means alone. Such projects are becoming increasingly attractive with the appearance of faster and more powerful quantitative techniques in both biological data acquisition and data storage, manipulation, and presentation. However, as is frequent in interdisciplinary research, the main hurdles are not within the mindset and techniques of each discipline but are usually encountered in attempting to meld the different disciplines together. Based upon our experience in applying mathematical methods to investigate how neural crest cells interact to form the enteric nervous system, we present our views on how to pursue biomathematical modeling projects, what difficulties to expect, and how to overcome, or at least survive, these hurdles. The main advice being: persevere.
Constitutive release of CPS1 in bile and its role as a protective cytokine during acute liver injury.
Apr 13, 2019   Proceedings Of The National Academy Of Sciences Of The United States Of America
Park MJ, D'Alecy LG, Anderson MA, Basrur V, Feng Y, Brady GF, Kim DI, Wu J, Nesvizhskii AI, Lahann J, Lukacs NW, Fontana RJ, Omary MB
Constitutive release of CPS1 in bile and its role as a protective cytokine during acute liver injury.
Apr 13, 2019
Proceedings Of The National Academy Of Sciences Of The United States Of America
Carbamoyl phosphate synthetase-1 (CPS1) is the major mitochondrial urea cycle enzyme in hepatocytes. It is released into mouse and human blood during acute liver injury, where is has a short half-life. The function of CPS1 in blood and the reason for its short half-life in serum are unknown. We show that CPS1 is released normally into mouse and human bile, and pathologically into blood during acute liver injury. Other cytoplasmic and mitochondrial urea cycle enzymes are also found in normal mouse bile. Serum, bile, and purified CPS1 manifest sedimentation properties that overlap with extracellular vesicles, due to the propensity of CPS1 to aggregate despite being released primarily as a soluble protein. During liver injury, CPS1 in blood is rapidly sequestered by monocytes, leading to monocyte M2-polarization and homing to the liver independent of its enzyme activity. Recombinant CPS1 (rCPS1), but not control r-transferrin, increases hepatic macrophage numbers and phagocytic activity. Notably, rCPS1 does not activate hepatic macrophages directly; rather, it activates bone marrow and circulating monocytes that then home to the liver. rCPS1 administration prevents mouse liver damage induced by Fas ligand or acetaminophen, but this protection is absent in macrophage-deficient mice. Moreover, rCPS1 protects from acetaminophen-induced liver injury even when given therapeutically after injury induction. In summary, CPS1 is normally found in bile but is released by hepatocytes into blood upon liver damage. We demonstrate a nonenzymatic function of CPS1 as an antiinflammatory protective cytokine during acute liver injury.
Genistein antagonizes gliadin-induced CFTR malfunction in models of celiac disease.
Apr 13, 2019   Aging
Esposito S, Villella VR, Ferrari E, Monzani R, Tosco A,   . . . . . .   , Romani L, Piacentini M, Raia V, Kroemer G, Maiuri L
Genistein antagonizes gliadin-induced CFTR malfunction in models of celiac disease.
Apr 13, 2019
Aging
In celiac disease (CD), an intolerance to dietary gluten/gliadin, antigenic gliadin peptides trigger an HLA-DQ2/DQ8-restricted adaptive Th1 immune response. Epithelial stress, induced by other non-antigenic gliadin peptides, is required for gliadin to become fully immunogenic. We found that cystic-fibrosis-transmembrane-conductance-regulator (CFTR) acts as membrane receptor for gliadin-derived peptide P31-43, as it binds to CFTR and impairs its channel function. P31-43-induced CFTR malfunction generates epithelial stress and intestinal inflammation. Maintaining CFTR in an active open conformation by the CFTR potentiators VX-770 (Ivacaftor) or Vrx-532, prevents P31-43 binding to CFTR and controls gliadin-induced manifestations. Here, we evaluated the possibility that the over-the-counter nutraceutical genistein, known to potentiate CFTR function, would allow to control gliadin-induced alterations. We demonstrated that pre-treatment with genistein prevented P31-43-induced CFTR malfunction and an epithelial stress response in Caco-2 cells. These effects were abrogated when the CFTR gene was knocked out by CRISP/Cas9 technology, indicating that genistein protects intestinal epithelial cells by potentiating CFTR function. Notably, genistein protected gliadin-sensitive mice from intestinal CFTR malfunction and gliadin-induced inflammation as it prevented gliadin-induced IFN-γ production by celiac peripheral-blood-mononuclear-cells (PBMC) cultured ex-vivo in the presence of P31-43-challenged Caco-2 cells. Our results indicate that natural compounds capable to increase CFTR channel gating might be used for the treatment of CD.
Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult-Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model.
Apr 17, 2019   Proteomics
Guergues J, Zhang P, Liu B, Stevens SM
Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult-Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model.
Apr 17, 2019
Proteomics
Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain which can limit their relevance to homeostatic function and disease-related neuroimmune responses in the adult brain. Recently, an adult mouse brain-derived microglial cell line was established; however, a comprehensive proteome dataset remains lacking. We here describe an optimization method for sensitive and rapid quantitative proteomic analysis of microglia that involves suspension trapping (S-Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (∼300K). Using a 2-hr gradient on a 75-cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer, 4,855 total proteins were identified where 4,698 of which were quantifiable by label-free quantitation with a median and average CV of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult-derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006. This article is protected by copyright. All rights reserved.
Proteomics and N-Glycoproteomics analysis of an extracellular matrix-based scaffold-human treated dentin matrix.
Apr 13, 2019   Journal Of Tissue Engineering And Regenerative Medicine
Li J, Yang H, Lu Q, Chen D, Zhou M, Kuang Y, Ying S, Song J
Proteomics and N-Glycoproteomics analysis of an extracellular matrix-based scaffold-human treated dentin matrix.
Apr 13, 2019
Journal Of Tissue Engineering And Regenerative Medicine
Extracellular matrix (ECM)-based biomaterials developed from mammalian tissues have been successfully used in preclinical and clinical tissue engineering applications. We have previously reported about the applicability of dentin-based scaffold, treated dentin matrix (TDM), for tooth root regeneration. However, TDM protein composition has not been characterized. Here, we used a shotgun proteomic strategy to profile human TDM proteome. N-glycoproteins were enriched by lectin affinity chromatography and identified by mass spectrometry. The total human TDM proteome was compared with the previously published human dentin proteome, and bioinformatics analysis were performed accordingly. In total, 708 proteins were identified by MS in human TDM, of which 208 were N-glycoproteins with 318 identified glycosylation sites. Collagens, proteoglycans, small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and growth factors, such as COL1A1, biglycan, dentin sialoprotein, transforming growth factor beta 1, were identified. Glycoproteins were enriched in "biological processes" Gene Ontology terms such as cellular process, biological regulation, response to stimulus, metabolic process, immune system process, and biological adhesion. Thus, our comprehensive study of the human TDM proteome revealed that dentin proteins are more heterogeneous than previously documented. Our findings provide clues for designing new biomaterials for tooth root regeneration and understanding dentin formation.
Quantitative Proteomics Data in the Public Domain: Challenges and Opportunities.
Apr 13, 2019   Methods In Molecular Biology (Clifton, N.J.)
Jarnuczak AF, Ternent T, Vizcaíno JA
Quantitative Proteomics Data in the Public Domain: Challenges and Opportunities.
Apr 13, 2019
Methods In Molecular Biology (Clifton, N.J.)
Mass spectrometry based proteomics is no longer only a qualitative discipline, and can be successfully employed to obtain a truly multidimensional view of the proteome. In particular, systematic protein expression profiling is now a routine part of many studies in the field and beyond. The large growth in the number of quantitative studies is accompanied by a trend to share publicly the associated analysis results and the underlying raw data. This trend, established and strongly supported by public repositories such as the PRIDE database at the European Bioinformatics Institute, opens up enormous possibilities to explore the data beyond the original publications, for instance by reusing, reanalyzing, and performing different flavors of meta-analysis studies. To help researchers and scientists realize about this potential, here we describe the mainstream public proteomics resources containing quantitative proteomics data, including the processed analysis results and/or the underlying raw data. We then present and discuss the most important points to consider when attempting to (re)use proteomics data in the public domain. We conclude by highlighting potential pitfalls of (re)using quantitative data and discuss some of our own experiences in this context.
Practical Integration of Multi-Run iTRAQ Data.
Apr 13, 2019   Methods In Molecular Biology (Clifton, N.J.)
Pascovici D, Song X, Wu J, Zaw T, Molloy M
Practical Integration of Multi-Run iTRAQ Data.
Apr 13, 2019
Methods In Molecular Biology (Clifton, N.J.)
In this chapter, we describe some of the approaches we employ in the analysis of iTRAQ data in our group, with an emphasis on practical issues that can occur in larger multi-run projects. Our pipeline starts with a well-established iTRAQ workflow, makes use of protein level quantitation using ProteinPilot, and continues either via a global analysis in the presence of a common reference, or by identifying pairwise comparisons of interest and applying a method taking the protein ratios and protein ratio confidence measures into consideration. Additionally we describe what issues can occur in the more subtle scenarios involving composite databases in multi-run situations, and an approach applicable in that setting.
Quantitative Analysis of Protein S-Acylation Site Dynamics Using Site-Specific Acyl-Biotin Exchange (ssABE).
Apr 13, 2019   Methods In Molecular Biology (Clifton, N.J.)
Woodley KT, Collins MO
Quantitative Analysis of Protein S-Acylation Site Dynamics Using Site-Specific Acyl-Biotin Exchange (ssABE).
Apr 13, 2019
Methods In Molecular Biology (Clifton, N.J.)
Protein S-acylation (palmitoylation) is a reversible lipid modification that is increasingly recognized as an important regulator of protein function, including membrane association, trafficking, and subcellular localization. Most proteomic methods to study palmitoylation allow characterization of putative palmitoylated proteins but do not permit identification of individual sites of palmitoylation. We have recently adapted the Acyl-Biotin Exchange (ABE) method that is routinely used for palmitoyl-proteome characterization, to permit global S-acylation site analysis. This site-specific ABE (ssABE) protocol, when combined with SILAC-based quantification, allows both the large-scale identification of palmitoylation sites and quantitative profiling of palmitoylation site changes. This approach enables palmitoylation to be studied at a systems level comparable to other more intensively studied post-translational modifications.
Quantitative Phosphoproteomic Using Titanium Dioxide Micro-Columns and Label-Free Quantitation.
Apr 13, 2019   Methods In Molecular Biology (Clifton, N.J.)
Barrios-Llerena ME, Le Bihan T
Quantitative Phosphoproteomic Using Titanium Dioxide Micro-Columns and Label-Free Quantitation.
Apr 13, 2019
Methods In Molecular Biology (Clifton, N.J.)
Phosphorylation events are important during cellular function. Analysis of phosphorylation in complex samples has been extensively studied using large-scale phosphopeptide enrichment methods. Quantitative analysis of the enriched phosphopeptides is subsequently performed using label-based methodologies (e.g., SILAC, iTRAQ, and others). Here we describe the protocol for the quantitative analysis of phosphopeptides, enriched with titanium dioxide micro-column, using an intensity-based label-free quantitation.
Next Generation Proteomics for Clinical Biomarker Detection Using SWATH-MS.
Apr 13, 2019   Methods In Molecular Biology (Clifton, N.J.)
Lin Q, Tan HT, Chung MCM
Next Generation Proteomics for Clinical Biomarker Detection Using SWATH-MS.
Apr 13, 2019
Methods In Molecular Biology (Clifton, N.J.)
The technology of "sequential windowed acquisition of all theoretical fragment ion spectra," known as SWATH-MS, is rapidly gaining popularity as a next generation proteomics technology for comprehensive proteome quantitation. In this chapter, we describe the use of SWATH-MS as a label-free quantitative technique in a proteomics study to identify novel serological biomarker for colorectal cancer. We compared the secreted glycoprotein profiles (glyco-secretomes) enriched from the colon adenocarcinoma cell line HCT-116 and its metastatic derivative, E1, and observed that laminin β-1 (LAMB1) was oversecreted in E1 cells. This novel oversecretion of LAMB1 was validated in colorectal cancer patient serum samples, and ROC analyses showed that LAMB1 performed better than carcinoembryonic antigen (CEA) as a clinical diagnostic biomarker for colorectal cancer. We focus here on the sample preparation methodology and data processing workflow for SWATH-MS studies.
Phylogenetic Studies.
Apr 13, 2019   Methods In Molecular Biology (Clifton, N.J.)
Kuhls K, Mauricio I
Phylogenetic Studies.
Apr 13, 2019
Methods In Molecular Biology (Clifton, N.J.)
Phylogenetics is an important component of the systems biology approach. Knowledge about evolution of the genus Leishmania is essential to understand various aspects of basic biology of these parasites, such as parasite-host or parasite-vector relationships, biogeography, or epidemiology. Here, we present a comprehensive guideline for performing phylogenetic studies based on DNA sequence data, but with principles that can be adapted to protein sequences or other molecular markers. It is presented as a compilation of the most commonly used genetic targets for phylogenetic studies of Leishmania, including their respective primers for amplification and references, as well as details of PCR assays. Guidelines are, then, presented to choose the best targets in relation to the types of samples under study. Finally, and importantly, instructions are given to obtain optimal sequences, alignments, and datasets for the subsequent data analysis and phylogenetic inference. Different bioinformatics methods and software for phylogenetic inference are presented and explained. This chapter aims to provide a compilation of methods and generic guidelines to conduct phylogenetics of Leishmania for nonspecialists.
Definitions and guidelines for research on antibiotic persistence.
Apr 13, 2019   Nature Reviews. Microbiology
Balaban NQ, Helaine S, Lewis K, Ackermann M, Aldridge B,   . . . . . .   , Storz G, Tan MW, Tenson T, Van Melderen L, Zinkernagel A
Definitions and guidelines for research on antibiotic persistence.
Apr 13, 2019
Nature Reviews. Microbiology
Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.
Integrated transcriptomic and proteomic analysis of human eccrine sweat glands identifies missing and novel proteins.
Apr 13, 2019   Molecular & Cellular Proteomics : MCP
Na CH, Sharma N, Madugundu AK, Chen R, Atalar Aksit M, Rosson GD, Cutting GR, Pandey A
Integrated transcriptomic and proteomic analysis of human eccrine sweat glands identifies missing and novel proteins.
Apr 13, 2019
Molecular & Cellular Proteomics : MCP
The eccrine sweat gland is an exocrine gland that is involved in the secretion of sweat for control of temperature. Malfunction of the sweat glands can result in disorders such as miliaria, hyperhidrosis and bromhidrosis. Understanding the transcriptome and proteome of sweat glands is important for understanding their physiology and role in diseases. However, no systematic transcriptome or proteome analysis of sweat glands has yet been reported. Here, we isolated eccrine sweat glands from human skin by microdissection and performed RNA-seq and proteome analysis. In total, ~138,000 transcripts and ~6,100 proteins were identified. Comparison of the RNA-seq data of eccrine sweat glands to other human tissues revealed the closest resemblance to the cortex region of kidneys. The proteome data showed enrichment of proteins involved in secretion, reabsorption, and wound healing. Importantly, protein level identification of the calcium ion channel TRPV4 suggests the importance of eccrine sweat glands in re-epithelialization of wounds and prevention of dehydration. We also identified 2 previously missing proteins from our analysis. Using a proteogenomic approach, we identified 7 peptides from 5 novel genes, which we validated using synthetic peptides. Most of the novel proteins were from short open reading frames (sORFs) suggesting that many sORFs still remain to be annotated in the human genome. This study presents the first integrated analysis of the transcriptome and proteome of the human eccrine sweat gland and would become a valuable resource for studying sweat glands in physiology and disease.
Proteomics in Neural Crest Cell Research.
Apr 12, 2019   Methods In Molecular Biology (Clifton, N.J.)
McCarthy P, Schwarz Q
Proteomics in Neural Crest Cell Research.
Apr 12, 2019
Methods In Molecular Biology (Clifton, N.J.)
Detailed investigation of the neural crest cell proteome has lingered behind that of the transcriptome due to the challenge of obtaining sufficient starting material for subsequent proteomic investigation. Compounded by the complexity of protein abundance, large number of posttranslational modifications, and the stochastic nature of proteomics approaches, little data so far exists describing the true neural crest cell proteome. However, recent advances in instrument sensitivity and recovery of material during sample preparation have alleviated many of these problems and make proteomics analysis an underutilized tool to study neural crest cell biology. Here we present a quantitative proteomics protocol for deep analysis of both whole proteome and posttranslational modifications and a separate protocol for ultrasensitive proteomic profiling from submicrogram amounts of protein.
YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of satellite cells.
Apr 13, 2019   The EMBO Journal
Chen F, Zhou J, Li Y, Zhao Y, Yuan J,   . . . . . .   , Cheung TH, Wu Z, Wong CC, Sun H, Wang H
YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of satellite cells.
Apr 13, 2019
The EMBO Journal
Skeletal muscle satellite cells (SCs) are adult muscle stem cells responsible for muscle regeneration after acute or chronic injuries. The lineage progression of quiescent SC toward activation, proliferation, and differentiation during the regeneration is orchestrated by cascades of transcription factors (TFs). Here, we elucidate the function of TF Yin Yang1 (YY1) in muscle regeneration. Muscle-specific deletion of YY1 in embryonic muscle progenitors leads to severe deformity of diaphragm muscle formation, thus neonatal death. Inducible deletion of YY1 in SC almost completely blocks the acute damage-induced muscle repair and exacerbates the chronic injury-induced dystrophic phenotype. Examination of SC revealed that YY1 loss results in cell-autonomous defect in activation and proliferation. Mechanistic search revealed that YY1 binds and represses mitochondrial gene expression. Simultaneously, it also stabilizes Hif1α protein and activates Hif1α-mediated glycolytic genes to facilitate a metabolic reprogramming toward glycolysis which is needed for SC proliferation. Altogether, our findings have identified YY1 as a key regulator of SC metabolic reprogramming through its dual roles in modulating both mitochondrial and glycolytic pathways.
Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.
Apr 13, 2019   Immunity
Zilionis R, Engblom C, Pfirschke C, Savova V, Zemmour D,   . . . . . .   , Tenen DG, Bueno R, Levantini E, Pittet MJ, Klein AM
Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.
Apr 13, 2019
Immunity
Tumor-infiltrating myeloid cells (TIMs) comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients' blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.
Intrinsically Disordered Proteins in Chronic Diseases.
Apr 13, 2019   Biomolecules
Kulkarni P, Uversky VN
Intrinsically Disordered Proteins in Chronic Diseases.
Apr 13, 2019
Biomolecules
t is now increasingly evident that a large fraction of the human proteome comprises proteins that, under physiological conditions, lack fixed, ordered 3D structures as a whole or have segments that are not likely to form a defined 3D structure [...].
Dynamical systems approaches to personalized medicine.
Apr 16, 2019   Current Opinion In Biotechnology
Davis JD, Kumbale CM, Zhang Q, Voit EO
Dynamical systems approaches to personalized medicine.
Apr 16, 2019
Current Opinion In Biotechnology
The complexity of the human body is a major roadblock to diagnosis and treatment of disease. Individuals may be diagnosed with the same disease but exhibit different biomarker profiles or physiological changes and, importantly, they may respond differently to the same risk factors and the same treatment. There is no doubt that computational methods of data analysis and interpretation must be developed for medicine to evolve from the traditional population-based approaches to personalized treatment strategies. We discuss how computational systems biology is contributing to this current evolution.
Beyond bulk: a review of single cell transcriptomics methodologies and applications.
Apr 16, 2019   Current Opinion In Biotechnology
Kulkarni A, Anderson AG, Merullo DP, Konopka G
Beyond bulk: a review of single cell transcriptomics methodologies and applications.
Apr 16, 2019
Current Opinion In Biotechnology
Single-cell RNA sequencing (scRNA-seq) is a promising approach to study the transcriptomes of individual cells in the brain and the central nervous system (CNS). This technology acts as a bridge between neuroscience, computational biology, and systems biology, enabling an unbiased and novel understanding of the cellular composition of the brain and CNS. Gene expression at the single cell resolution is often noisy, sparse, and high-dimensional, creating challenges for computational analysis of such data. In this review, we overview fundamental sample preparation and data analysis processes of scRNA-seq and provide a comparative perspective for analyzing and visualizing these data.
Proteomic manifestations of genetic defects in autosomal recessive congenital ichthyosis.
Apr 12, 2019   Journal Of Proteomics
Karim N, Durbin-Johnson B, Rocke DM, Salemi M, Phinney BS, Naeem M, Rice RH
Proteomic manifestations of genetic defects in autosomal recessive congenital ichthyosis.
Apr 12, 2019
Journal Of Proteomics
Numerous genetic conditions give rise to a scaly skin phenotype as a result of impaired barrier function. Present work investigates the degree to which the departure from normal of ichthyosis corneocytes on the skin surface depends upon the basic defect as judged by proteomic profiling. Analyzing autosomal recessive congenital ichthyosis arising from defects in the genes PNPLA1, SDR9C7 and TGM1 revealed that profiles of PNPLA1 samples displayed the greatest degree of departure from normal control epidermis, with SDR9C7 samples nearly as divergent, and TGM1 the least divergent. Although the profiles were distinctive, each displaying a set of altered protein levels, they exhibited alterations in 20 proteins in common, of which 15 were expressed consistently at higher and 5 at lower levels. Departure from the normal profile was examined at three different anatomic sites (forearm, forehead, leg). Reflecting that the normal protein profile differed at these sites, comparing profiles from afflicted subjects revealed that the degree of alteration in profile was site-dependent. These results suggest proteomic profiling can provide a quantitative measure of departure from the normal state of epidermis. Further development may help characterize consequences of the genetic defects, including perturbation of signaling pathways, and supplement visual evaluation of treatment. SIGNIFICANCE: ARCI are rare cornification disorders caused by mutations in at least 14 different genes leading to perturbed metabolism and organization of constituent biomolecules of cornified envelope. The phenotypic manifestations of the disorder vary among individuals with the same as well as different genetic defect and even at different anatomic sites within the same individual. The present study investigates the proteomic disturbances at three anatomic sites in patients carrying mutations in three different genes. Our findings provide a basis for elucidating genotype to proteome relationships for ARCI, further investigation of which may help to delineate the underlying pathways as well as to identify new drug targets.
Combined Analysis of Metabolomes, Proteomes, and Transcriptomes of HCV-infected Cells and Liver to Identify Pathways Associated With Disease Development.
Apr 12, 2019   Gastroenterology
Lupberger J, Croonenborghs T, Roca Suarez AA, Van Renne N, Jühling F,   . . . . . .   , Gevaert O, Zeisel MB, Hoshida Y, Pochet N, Baumert TF
Combined Analysis of Metabolomes, Proteomes, and Transcriptomes of HCV-infected Cells and Liver to Identify Pathways Associated With Disease Development.
Apr 12, 2019
Gastroenterology
BACKGROUND & AIMS: The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes. METHODS: Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA-Seq, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA-seq and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsies from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function. RESULTS: We quantified 21,950 mRNAs and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of mRNAs, but not proteins, that regulate the innate immune response-we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate beta-oxidation of very long-chain fatty acids (VLCFAs); we found intracellular accumulation of VLCFAs in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of mRNAs and proteins associated with peroxisome function, indication perturbation of peroxisomes. We associated defects in peroxisome function with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients. CONCLUSIONS: We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in expression of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.
Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size.
Apr 12, 2019   PLoS Computational Biology
Rosenbloom DIS, Bacchetti P, Stone M, Deng X, Bosch RJ,   . . . . . .   , Lai J, Sobolewski MD, Kulpa DA, Busch MP, Reservoir Assay Validation and Evaluation Network (RAVEN) Study Group
Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size.
Apr 12, 2019
PLoS Computational Biology
Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.
SolidBin: Improving Metagenome Binning with Semi-supervised Normalized Cut.
Apr 12, 2019   Bioinformatics (Oxford, England)
Wang Z, Wang Z, Lu YY, Sun F, Zhu S
SolidBin: Improving Metagenome Binning with Semi-supervised Normalized Cut.
Apr 12, 2019
Bioinformatics (Oxford, England)
MOTIVATION: Metagenomic contig binning is an important computational problem in metagenomic research, which aims to cluster contigs from the same genome into the same group. Unlike classical clustering problem, contig binning can utilize known relationships among some of the contigs or the taxonomic identity of some contigs. However, the current state-of-the-art contig binning methods do not make full use of the additional biological information except the coverage and sequence composition of the contigs. RESULTS: We developed a novel contig binning method, SolidBin (Semi-supervised Spectral Normalized Cut for Binning), based on semi-supervised spectral clustering. Using sequence feature similarity and/or additional biological information, such as the reliable taxonomy assignments of some contigs, SolidBin constructs two types of prior information: must-link and cannot-link constraints. Must-link constraints mean that the pair of contigs should be clustered into the same group, while cannot-link constraints mean that the pair of contigs should be clustered in different groups. These constraints are then integrated into a classical spectral clustering approach, normalized cut (NCut), for improved contig binning. The performance of SolidBin is compared with five state-of-the-art genome binners, CONCOCT, COCACOLA, MaxBin, MetaBAT and BMC3C on five next-generation sequencing (NGS) benchmark datasets including simulated multi- and single-sample datasets and real multi-sample datasets. The experimental results show that, SolidBin has achieved the best performance in terms of F-score, ARI and NMI, especially while using the real datasets and the single sample dataset. AVAILABILITY: https://github.com/sufforest/SolidBin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
An immunosuppressive peptide from the horsefly inhibits inflammation by repressing macrophage maturation and phagocytosis.
Apr 12, 2019   Journal Of Cellular Biochemistry
Chen R, Yuan L, Cao N, Li P, Chen H, Zhou J, Hao X, Liu T, Yang WH, Cui S, Yan X
An immunosuppressive peptide from the horsefly inhibits inflammation by repressing macrophage maturation and phagocytosis.
Apr 12, 2019
Journal Of Cellular Biochemistry
Ectoparasites repress host immune responses while they obtain nutrition from their hosts. Understanding the immunosuppressive mechanisms between ectoparasites and their hosts will provide new strategies to develop potential immunosuppressive drugs against immune disorder diseases. Previously, we have discovered that a small peptide, immunoregulin HA, from the horsefly (Hybomitra atriperoides) may play an immunosuppressive role in rat splenocytes. However, the targeting cells and detailed mechanisms of immunoregulin HA in immunosuppressive reactions are not well defined. Here, we show that immunoregulin HA reduces the secretion of proinflammatory cytokines upon lipopolysaccharide (LPS) stimulation. Interestingly, we discover that the major cytokines repressed by immunoregulin HA are secreted by macrophages, rather than by T cells. Furthermore, immunoregulin HA inhibits macrophage maturation and phagocytosis. Mechanically, the activations of c-JUN N-terminal kinase and extracellular signal-regulated kinase upon LPS stimulation are decreased by immunoregulin HA. Consistently, immunoregulin HA treatment exhibits an anti-inflammatory activity in a mouse model of adjuvant-induced paw inflammation. Taken together, our data reveal that immunoregulin HA conducts the anti-inflammatory activity by blocking macrophage functions.
A patient with homozygous nonsense variants in two Leigh syndrome disease genes: Distinguishing a dual diagnosis from a hypomorphic protein-truncating variant.
Apr 13, 2019   Human Mutation
Lake NJ, Formosa LE, Stroud DA, Ryan MT, Calvo SE, Mootha VK, Morar B, Procopis PG, Christodoulou J, Compton AG, Thorburn DR
A patient with homozygous nonsense variants in two Leigh syndrome disease genes: Distinguishing a dual diagnosis from a hypomorphic protein-truncating variant.
Apr 13, 2019
Human Mutation
Leigh syndrome is a mitochondrial disease caused by pathogenic variants in over 85 genes. Whole exome sequencing of a patient with Leigh-like syndrome identified homozygous protein-truncating variants in two genes associated with Leigh syndrome; a reported pathogenic variant in PDHX (NP_003468.2:p.(Arg446*)), and an uncharacterized variant in complex I (CI) assembly factor TIMMDC1 (NP_057673.2:p.(Arg225*)). The TIMMDC1 variant was predicted to truncate 61 amino acids at the C-terminus and functional studies demonstrated a hypomorphic impact of the variant on CI assembly. However, the mutant protein could still rescue CI assembly in TIMMDC1 knockout cells and the patient's clinical phenotype was not clearly distinct from that of other patients with the same PDHX defect. Our data suggest that the hypomorphic effect of the TIMMDC1 protein-truncating variant does not constitute a dual diagnosis in this individual. We recommend cautious assessment of variants in the C-terminus of TIMMDC1 and emphasize the need to consider the caveats detailed within the American College of Medical Genetics and Genomics (ACMG) criteria when assessing variants.

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