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A digital microfluidic system for serological immunoassays in remote settings.

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A digital microfluidic system for serological immunoassays in remote settings.

Sci Transl Med. 2018 Apr 25;10(438):

Authors: Ng AHC, Fobel R, Fobel C, Lamanna J, Rackus DG, Summers A, Dixon C, Dryden MDM, Lam C, Ho M, Mufti NS, Lee V, Asri MAM, Sykes EA, Chamberlain MD, Joseph R, Ope M, Scobie HM, Knipes A, Rota PA, Marano N, Chege PM, Njuguna M, Nzunza R, Kisangau N, Kiogora J, Karuingi M, Burton JW, Borus P, Lam E, Wheeler AR

Abstract
Serosurveys are useful for assessing population susceptibility to vaccine-preventable disease outbreaks. Although at-risk populations in remote areas could benefit from this type of information, they face several logistical barriers to implementation, such as lack of access to centralized laboratories, cold storage, and transport of samples. We describe a potential solution: a compact and portable, field-deployable, point-of-care system relying on digital microfluidics that can rapidly test a small volume of capillary blood for disease-specific antibodies. This system uses inexpensive, inkjet-printed digital microfluidic cartridges together with an integrated instrument to perform enzyme-linked immunosorbent assays (ELISAs). We performed a field validation of the system's analytical performance at Kakuma refugee camp, a remote setting in northwestern Kenya, where we tested children aged 9 to 59 months and caregivers for measles and rubella immunoglobulin G (IgG). The IgG assays were determined to have sensitivities of 86% [95% confidence interval (CI), 79 to 91% (measles)] and 81% [95% CI, 73 to 88% (rubella)] and specificities of 80% [95% CI, 49 to 94% (measles)] and 91% [95% CI, 76 to 97% (rubella)] (measles, n = 140; rubella, n = 135) compared with reference tests (measles IgG and rubella IgG ELISAs from Siemens Enzygnost) conducted in a centralized laboratory. These results demonstrate a potential role for this point-of-care system in global serological surveillance, particularly in remote areas with limited access to centralized laboratories.

PMID: 29695457 [PubMed - in process]



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Disruption of protein function by pathogenic mutations: common and uncommon mechanisms.

Disruption of protein function by pathogenic mutations: common and uncommon mechanisms.

Biochem Cell Biol. 2018 Apr 25;:

Authors: Taipale M

Abstract
Mutations in protein coding regions underlie almost all Mendelian disorders, drive tumorigenesis, and contribute to susceptibility to common diseases. Despite the great diversity of diseases that are caused by coding mutations, the cellular processes that affect - and are affected by - pathogenic variants at the molecular level are fundamentally conserved. Experimental and computational approaches have revealed that a substantial fraction of disease mutations are not simple loss-of-function alleles. Rather, these pathogenic variants disrupt protein function in more subtle ways by tuning protein folding pathways, altering subcellular trafficking, interrupting signaling cascades, and rewiring highly connected interaction networks. Focusing mainly on Mendelian disorders, this review discusses the common mechanisms by which deleterious mutations disrupt protein function and how these disruptions can be exploited in the development of novel therapies.

PMID: 29693415 [PubMed - as supplied by publisher]



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The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression.

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The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression.

Cell. 2018 Apr 14;:

Authors: Espiritu SMG, Liu LY, Rubanova Y, Bhandari V, Holgersen EM, Szyca LM, Fox NS, Chua MLK, Yamaguchi TN, Heisler LE, Livingstone J, Wintersinger J, Yousif F, Lalonde E, Rouette A, Salcedo A, Houlahan KE, Li CH, Huang V, Fraser M, van der Kwast T, Morris QD, Bristow RG, Boutros PC

Abstract
The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance.

PMID: 29681457 [PubMed - as supplied by publisher]



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Chemically controlled aggregation of pluripotent stem cells.

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Chemically controlled aggregation of pluripotent stem cells.

Biotechnol Bioeng. 2018 Apr 21;:

Authors: Lipsitz YY, Tonge P, Zandstra PW

Abstract
Heterogeneity in pluripotent stem cell (PSC) aggregation leads to variability in mass transfer and signaling gradients between aggregates, which results in heterogeneous differentiation and therefore variability in product quality and yield. We have characterized a chemical based method to control aggregate size within a specific, tunable range with low heterogeneity, thereby reducing process variability in PSC expansion. This method enables controlled, scalable, stirred suspension based manufacturing of PSC cultures which are critical for the translation of regenerative medicine strategies to clinical products. This article is protected by copyright. All rights reserved.

PMID: 29679475 [PubMed - as supplied by publisher]



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Donated chemical probes for open science.

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Donated chemical probes for open science.

Elife. 2018 Apr 20;7:

Authors: Müller S, Ackloo S, Arrowsmith CH, Bauser M, Baryza JL, Blagg J, Böttcher J, Bountra C, Brown PJ, Bunnage ME, Carter AJ, Damerell D, Dötsch V, Drewry DH, Edwards AM, Edwards J, Elkins JM, Fischer C, Frye SV, Gollner A, Grimshaw CE, IJzerman A, Hanke T, Hartung IV, Hitchcock S, Howe T, Hughes TV, Laufer S, Li VM, Liras S, Marsden BD, Matsui H, Mathias J, O'Hagan RC, Owen DR, Pande V, Rauh D, Rosenberg SH, Roth BL, Schneider NS, Scholten C, Singh Saikatendu K, Simeonov A, Takizawa M, Tse C, Thompson PR, Treiber DK, Viana AY, Wells CI, Willson TM, Zuercher WJ, Knapp S, Mueller-Fahrnow A

Abstract
Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (<ext-link ext-link-type="uri" xlink:href="https://openscienceprobes.sgc-frankfurt.de">https://openscienceprobes.sgc-frankfurt.de</ext-link><ext-link ext-link-type="uri" xlink:href="https://openscienceprobes.sgc-frankfurt.de/">/</ext-link>). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.

PMID: 29676732 [PubMed - in process]



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Systematic analysis of complex genetic interactions.

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Systematic analysis of complex genetic interactions.

Science. 2018 Apr 20;360(6386):

Authors: Kuzmin E, VanderSluis B, Wang W, Tan G, Deshpande R, Chen Y, Usaj M, Balint A, Mattiazzi Usaj M, van Leeuwen J, Koch EN, Pons C, Dagilis AJ, Pryszlak M, Wang JZY, Hanchard J, Riggi M, Xu K, Heydari H, San Luis BJ, Shuteriqi E, Zhu H, Van Dyk N, Sharifpoor S, Costanzo M, Loewith R, Caudy A, Bolnick D, Brown GW, Andrews BJ, Boone C, Myers CL

Abstract
To systematically explore complex genetic interactions, we constructed ~200,000 yeast triple mutants and scored negative trigenic interactions. We selected double-mutant query genes across a broad spectrum of biological processes, spanning a range of quantitative features of the global digenic interaction network and tested for a genetic interaction with a third mutation. Trigenic interactions often occurred among functionally related genes, and essential genes were hubs on the trigenic network. Despite their functional enrichment, trigenic interactions tended to link genes in distant bioprocesses and displayed a weaker magnitude than digenic interactions. We estimate that the global trigenic interaction network is ~100 times as large as the global digenic network, highlighting the potential for complex genetic interactions to affect the biology of inheritance, including the genotype-to-phenotype relationship.

PMID: 29674565 [PubMed - in process]



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Systems analysis of the genetic interaction network of yeast molecular chaperones.

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Systems analysis of the genetic interaction network of yeast molecular chaperones.

Mol Omics. 2018 Apr 16;14(2):82-94

Authors: Rizzolo K, Kumar A, Kakihara Y, Phanse S, Minic Z, Snider J, Stagljar I, Zilles S, Babu M, Houry WA

Abstract
Molecular chaperones are typically promiscuous interacting proteins that function globally in the cell to maintain protein homeostasis. Recently, we had carried out experiments that elucidated a comprehensive interaction network for the core 67 chaperones and 15 cochaperones in the budding yeast Saccharomyces cerevisiae [Rizzolo et al., Cell Rep., 2017, 20, 2735-2748]. Here, the genetic (i.e. epistatic) interaction network obtained for chaperones was further analyzed, revealing that the global topological parameters of the resulting network have a more central role in mediating interactions in comparison to the rest of the proteins in the cell. Most notably, we observed Hsp10, Hsp70 Ssz1 chaperone, and Hsp90 cochaperone Cdc37 to be the main drivers of the network architecture. Systematic analysis on the physicochemical properties for all chaperone interactors further revealed the presence of preferential domains and folds that are highly interactive with chaperones such as the WD40 repeat domain. Further analysis with established cellular complexes revealed the involvement of R2TP chaperone in quaternary structure formation. Our results thus provide a global overview of the chaperone network properties in yeast, expanding our understanding of their functional diversity and their role in protein homeostasis.

PMID: 29659649 [PubMed]



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Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.

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Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.

Mol Cell. 2018 Apr 11;:

Authors: Ordureau A, Paulo JA, Zhang W, Ahfeldt T, Zhang J, Cohn EF, Hou Z, Heo JM, Rubin LL, Sidhu SS, Gygi SP, Harper JW

Abstract
Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.

PMID: 29656925 [PubMed - as supplied by publisher]



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Inference of the Human Polyadenylation Code.

Inference of the Human Polyadenylation Code.

Bioinformatics. 2018 Apr 10;:

Authors: Leung MKK, Delong A, Frey BJ

Abstract
Motivation: Processing of transcripts at the 3'-end involves cleavage at a polyadenylation site followed by the addition of a poly(A)-tail. By selecting which site is cleaved, the process of alternative polyadenylation enables genes to produce transcript isoforms with different 3'-ends. To facilitate the identification and treatment of disease-causing mutations that affect polyadenylation and to understand the sequence determinants underlying this regulatory process, a computational model that can accurately predict polyadenylation patterns from genomic features is desirable.
Results: Previous works have focused on identifying candidate polyadenylation sites and classifying tissue-specific sites. By training on how multiple sites in genes are competitively selected for polyadenylation from 3'-end sequencing data, we developed a deep learning model that can predict the tissue-specific strength of a polyadenylation site in the 3' untranslated region of the human genome given only its genomic sequence. We demonstrate the model's broad utility on multiple tasks, without any application-specific training. The model can be used to predict which polyadenylation site is more likely to be selected in genes with multiple sites. It can be used to scan the 3' untranslated region to find candidate polyadenylation sites. It can be used to classify the pathogenicity of variants near annotated polyadenylation sites in ClinVar. It can also be used to anticipate the effect of antisense oligonucleotide experiments to redirect polyadenylation. We provide analysis on how different features affect the model's predictive performance and a method to identify sensitive regions of the genome at the single-based resolution that can affect polyadenylation regulation.
Contact: frey@psi.toronto.edu.
Supplementary information: Supplementary data are available at Bioinformatics online.

PMID: 29648582 [PubMed - as supplied by publisher]



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Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma.

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Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma.

N Engl J Med. 2018 04 12;378(15):1396-1407

Authors: Schmitz R, Wright GW, Huang DW, Johnson CA, Phelan JD, Wang JQ, Roulland S, Kasbekar M, Young RM, Shaffer AL, Hodson DJ, Xiao W, Yu X, Yang Y, Zhao H, Xu W, Liu X, Zhou B, Du W, Chan WC, Jaffe ES, Gascoyne RD, Connors JM, Campo E, Lopez-Guillermo A, Rosenwald A, Ott G, Delabie J, Rimsza LM, Tay Kuang Wei K, Zelenetz AD, Leonard JP, Bartlett NL, Tran B, Shetty J, Zhao Y, Soppet DR, Pittaluga S, Wilson WH, Staudt LM

Abstract
BACKGROUND: Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics.
METHODS: We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations.
RESULTS: We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition.
CONCLUSIONS: We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).

PMID: 29641966 [PubMed - indexed for MEDLINE]



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