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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 55,165 bioRxiv papers from 254,492 authors.

Most downloaded bioRxiv papers, since beginning of last month

51,791 results found. For more information, click each entry to expand.

301: CRISPR adenine and cytosine base editors with reduced RNA off-target activities
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Posted to bioRxiv 09 May 2019

CRISPR adenine and cytosine base editors with reduced RNA off-target activities
319 downloads molecular biology

Julian Grunewald, Ronghao Zhou, Sowmya Iyer, Caleb A Lareau, Sara P. Garcia, Martin J Aryee, J. Keith Joung

CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described enhanced A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.

302: Pre-existent adult sox10+ cardiomyocytes contribute to myocardial regeneration in the zebrafish
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Posted to bioRxiv 07 Jun 2019

Pre-existent adult sox10+ cardiomyocytes contribute to myocardial regeneration in the zebrafish
319 downloads developmental biology

Marcos Sande-Melón, Inês J Marques, María Galardi-Castilla, Xavier Langa, María Pérez-López, Marius Botos, Gabriela Martínez-Guzmán, David Miguel Ferreira-Francisco, Dinko Pavlinic, Vladimir Benes, Remy Bruggmann, Nadia Mercader

During heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from pre-existing ones. It is unclear whether the heart is comprised of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identified a subset of pre-existent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded massively after cryoinjury. Genetic ablation of sox10+ cardiomyocytes severely impaired cardiac regeneration revealing that they play a crucial role for heart regeneration.

303: Ultrastructural details of mammalian chromosome architecture
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Posted to bioRxiv 17 May 2019

Ultrastructural details of mammalian chromosome architecture
318 downloads genomics

Nils Krietenstein, Sameer Abraham, Sergey Venev, Nezar Abdennur, Johan Gibcus, Tsung-Han Hsieh, Krishna Mohan Parsi, Liyan Yang, Rene Maehr, Leonid Mirny, Job Dekker, Oliver Rando

Over the past decade, 3C-related methods, complemented by increasingly detailed microscopic views of the nucleus, have provided unprecedented insights into chromosome folding in vivo. Here, to overcome the resolution limits inherent to the majority of genome-wide chromosome architecture mapping studies, we extend a recently-developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human embryonic stem cells and fibroblasts. Micro-C maps robustly capture well-described features of mammalian chromosome folding including A/B compartment organization, topologically associating domains (TADs), and cis interaction peaks anchored at CTCF binding sites, while also providing a detailed 1-dimensional map of nucleosome positioning and phasing genome-wide. Compared to high-resolution in situ Hi-C, Micro-C exhibits substantially improved signal-to-noise with an order of magnitude greater dynamic range, enabling not only localization of domain boundaries with single-nucleosome accuracy, but also resolving more than 20,000 additional looping interaction peaks in each cell type. Intriguingly, many of these newly-identified peaks are localized along stripe patterns and form transitive grids, consistent with their anchors being pause sites impeding the process of cohesin-dependent loop extrusion. Together, our analyses provide the highest resolution maps of chromosome folding in human cells to date, and provide a valuable resource for studies of chromosome folding mechanisms.

304: Minor allele frequency thresholds strongly affect population structure inference with genomic datasets
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Posted to bioRxiv 14 Sep 2017

Minor allele frequency thresholds strongly affect population structure inference with genomic datasets
318 downloads genomics

Ethan B. Linck, C. J. Battey

Across the genome, the effects of different evolutionary processes and historical events can result in different classes of genetic variants (or alleles) characterized by their relative frequency in a given population. As a result, population genetic inference can be strongly affected by biases in laboratory and bioinformatics treatments that affect the site frequency spectrum, or SFS. Yet despite the widespread use of reduced-representation genomic datasets with nonmodel organisms, the potential consequences of these biases for downstream analyses remain poorly examined. Here, we assess the influence of minor allele frequency (MAF) thresholds implemented during variant detection on inference of population structure. We use simulated and empirical datasets to evaluate the effect of MAF thresholds on the ability to discriminate among populations and quantify admixture with both model-based and non-model-based clustering methods. We find model-based inference of population structure is highly sensitive to choice of MAF, and may be confounded by either including singletons or excluding all rare alleles. In contrast, non-model-based clustering is largely robust to MAF choice. Our results suggest that model-based inference of population structure can fail due to either natural demographic processes or assembly artifacts, with broad consequences for phylogeographic and population genetic studies using NGS data. We propose a simple hypothesis to explain this behavior and recommend a set of best practices for researchers seeking to describe population structure using reduced-representation libraries.

305: c-Jun Overexpressing CAR-T Cells are Exhaustion-Resistant and Mediate Enhanced Antitumor Activity
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Posted to bioRxiv 29 May 2019

c-Jun Overexpressing CAR-T Cells are Exhaustion-Resistant and Mediate Enhanced Antitumor Activity
318 downloads immunology

Rachel Christina Lynn, Evan W Weber, David Gennert, Elena Sotillo, Peng Xu, Zinaida Good, Hima Anbunathan, Robert Jones, Victor Tieu, Jeffrey Granja, Charles DeBourcy, Robbie Majzner, Ansuman Satpathy, Stephen R. Quake, Howard Chang, Crystal L Mackall

CAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of bZIP and IRF transcription factors that have been implicated in driving exhaustion. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in five different in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.

306: Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis
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Posted to bioRxiv 21 Jun 2019

Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis
317 downloads cell biology

Matthew Akamatsu, Ritvik Vasan, Daniel Serwas, Michael Ferrin, Padmini Rangamani, David G. Drubin

Force generation due to actin assembly is a fundamental aspect of membrane sculpting for many essential processes. In this work, we use a multiscale computational model constrained by experimental measurements to show that a minimal branched actin network is sufficient to internalize endocytic pits against physiological membrane tension. A parameter sweep identified the number of Arp2/3 complexes as particularly important for robust internalization, which prompted the development of a molecule-counting method in live mammalian cells. Using this method, we found that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Our simulations also revealed that actin networks self-organize in a radial branched array with barbed filament ends oriented to grow toward the base of the pit, and that the distribution of linker proteins around the endocytic pit is critical for this organization. Surprisingly, our model predicted that long actin filaments bend from their attachment sites in the coat to the base of the pit and store elastic energy that can be harnessed to drive endocytosis. This prediction was validated using cryo-electron tomography on cells, which revealed the presence of bent actin filaments along the endocytic site. Furthermore, we predict that under elevated membrane tension, the self-organized actin network directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, our study reveals that spatially constrained actin filament assembly utilizes an adaptive mechanism that enables endocytosis under varying physical constraints.

307: Influence of genetic variants on gene expression in human pancreatic islets – implications for type 2 diabetes
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Posted to bioRxiv 31 May 2019

Influence of genetic variants on gene expression in human pancreatic islets – implications for type 2 diabetes
316 downloads genomics

Ana Viñuela, Arushi Varshney, Martijn van de Bunt, Rashmi B. Prasad, Olof B Asplund, Amanda Bennett, Michael Boehnke, Andrew Anand Brown, Michael R. Erdos, João Fadista, Ola Hansson, Gad Hatem, Cédric Howald, Apoorva K Iyengar, Paul Johnson, Ulrika Krus, Patrick E MacDonald, Anubha Mahajan, Jocelyn E Manning Fox, Narisu Narisu, Vibe Nylander, Peter Orchard, Nikolay Oskolkov, Nikolaos I Panousis, Anthony Payne, Michael L. Stitzel, Swarooparani Vadlamudi, Ryan Welch, Francis S. Collins, Karen L Mohlke, Anna Gloyn, Laura J. Scott, Emmanouil Dermitzakis, Leif L Groop, Stephen C.J. Parker, Mark I McCarthy

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, many key tissues and cell-types required for appropriate functional inference are absent from large-scale resources such as ENCODE and GTEx. We explored the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using RNA-Seq and genotyping data from 420 islet donors. We find: (a) eQTLs have a variable replication rate across the 44 GTEx tissues (<73%), indicating that our study captured islet-specific cis-eQTL signals; (b) islet eQTL signals show marked overlap with islet epigenome annotation, though eQTL effect size is reduced in the stretch enhancers most strongly implicated in GWAS signal location; (c) selective enrichment of islet eQTL overlap with the subset of T2D variants implicated in islet dysfunction; and (d) colocalization between islet eQTLs and variants influencing T2D or related glycemic traits, delivering candidate effector transcripts at 23 loci, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in tissues of greatest disease-relevance while expanding our mechanistic insights into complex traits association loci activity with an expanded list of putative transcripts implicated in T2D development.

308: Multi-omics profiling establishes the polypharmacology of FDA Approved CDK4/6 inhibitors and the potential for differential clinical activity
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Posted to bioRxiv 07 Nov 2017

Multi-omics profiling establishes the polypharmacology of FDA Approved CDK4/6 inhibitors and the potential for differential clinical activity
312 downloads cancer biology

Marc Hafner, Caitlin E. Mills, Kartik Subramanian, Chen Chen, Mirra Chung, Sarah A. Boswell, Robert A. Everley, Changchang Liu, Charlotte S. Walmsley, Dejan Juric, Peter Sorger

The target profiles of many drugs are established early in their development and are not systematically revisited at the time of FDA approval. Thus, it is often unclear whether therapeutics with the same nominal targets but different chemical structures are functionally equivalent. In this paper we use five different phenotypic and biochemical assays to compare approved inhibitors of cyclin-dependent kinases 4/6 - collectively regarded as breakthroughs in the treatment of hormone receptor-positive breast cancer. We find that transcriptional, proteomic and phenotypic changes induced by palbociclib, ribociclib, and abemaciclib differ significantly; abemaciclib in particular has advantageous activities partially overlapping those of alvocidib, an older polyselective CDK inhibitor. In cells and mice, abemaciclib inhibits kinases other than CDK4/6 including CDK2/Cyclin A/E - implicated in resistance to CDK4/6 inhibition - and CDK1/Cyclin B. The multi-faceted experimental and computational approaches described here therefore uncover under-appreciated differences in CDK4/6 inhibitor activities with potential importance in treating human patients.

309: Preparation of Organ Scaffolds by Decellularization of Pancreas and Re-functionalization
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Posted to bioRxiv 07 Jan 2019

Preparation of Organ Scaffolds by Decellularization of Pancreas and Re-functionalization
311 downloads cell biology

Uday Chandrika K, Rekha Tripathi, Avinash Raj T, Sairam N, Vasundhara Kamineni Parlikar, Swami VB, Nandini Rangaraj, Mahesh Kumar Jerald, Shashi Singh

Extracellular matrix of each tissue is unique in composition, architecture and finer details that support the very identity of the organ by regulating the status/character of the cells within it. Tissue engineering centers around creating a niche similar to the natural one, with a purpose of developing an organ/oid. In this study, whole organ decellularization of pancreas was attempted followed by reseeding it with adult mesenchymal stem cells. Decellularization completely removes cells leaving behind extracellular matrix rich scaffold. After reseeding, mesenchymal stem cells differentiate into pancreas specific cells. Upon transplantation of recellularized pancreas in streptozotocin induced diabetic mice, this organ was capable of restoring its histomorphology and normal functioning. Restoration of endocrine islets, the exocrine acinar region, and vascular network was seen in transplanted pancreas. The entire process of functional recovery took about 20 days when the mice demonstrated glucoregulation, though none achieved gluconormalization. Transplanted mice upon feeding show insulin and c-peptide in circulation. This process demonstrates that natural scaffolds of soft organs can be refunctionalized using recipients cells to counter immune problems arising due to organ transplantation.

310: Automated cryo-EM sample preparation by pin-printing and jet vitrification
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Posted to bioRxiv 28 May 2019

Automated cryo-EM sample preparation by pin-printing and jet vitrification
311 downloads biophysics

Raimond B.G. Ravelli, Frank J.T. Nijpels, Rene J.M. Henderikx, Giulia Weissenberger, Sanne Thewessem, Abril Gijsbers, Bart W.A.M.M. Beulen, Carmen Lopez-Iglesias, Peter Peters

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide controlled and efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving 4 standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

311: Teaching R in the undergraduate ecology classroom: approaches, lessons learned, and recommendations
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Posted to bioRxiv 11 Jun 2019

Teaching R in the undergraduate ecology classroom: approaches, lessons learned, and recommendations
310 downloads scientific communication and education

Linda A Auker, Erika L Barthelmess

Ecology requires training in data management and analysis. In this paper, we present data from the last 10 years demonstrating the increase in the use of R, an open-source programming environment, in ecology and its prevalence as a required skill in job descriptions. Because of its transparent and flexible nature, R is increasingly used for data management and analysis in the field of ecology. Consequently, job postings targeting candidates with a bachelor's degree and a required knowledge of R have increased over the past ten years. We discuss our experiences teaching undergraduates R in two advanced ecology classes using different approaches. One approach, in a course with a field lab, focused on collecting, cleaning, and preparing data for analysis. The other approach, in a course without a field lab, focused on analyzing existing data sets and applying the results to content discussed in the lecture portion of the course. Our experiences determined that each approach had strengths and weaknesses. We recommend that above all, instructors of ecology and related subjects should be encouraged to include R in their coursework. Furthermore, instructors should be aware of the following: learning R is a separate skill from learning statistics; writing R assignments is a significant time sink for course preparation; and, there is a tradeoff between teaching R and teaching content. Determining how one's course fits into the curriculum and identifying resources outside of the classroom for students' continued practice will ensure that R training is successful and will extend beyond a one-semester course.

312: Long-Term Effects of a Novel Continuous Remote Care Intervention Including Nutritional Ketosis for the Management of Type 2 Diabetes: A 2-year Non-randomized Clinical Trial.
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Posted to bioRxiv 28 Nov 2018

Long-Term Effects of a Novel Continuous Remote Care Intervention Including Nutritional Ketosis for the Management of Type 2 Diabetes: A 2-year Non-randomized Clinical Trial.
309 downloads clinical trials

Shaminie J Athinarayanan, Rebecca N Adams, Sarah J Hallberg, Amy L McKenzie, Nasir H Bhanpuri, Wayne W Campbell, Jeff S Volek, Stephen D Phinney, James P McCarter

OBJECTIVE: Studies on long-term sustainability of low-carbohydrate approaches to treat diabetes are limited. We aim to assess the effects of a continuous care intervention (CCI) on retention, glycemic control, weight, body composition, cardiovascular, liver, kidney, thyroid, inflammatory markers, diabetes medication usage and disease outcomes at 2 years in adults with type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: An open label, non-randomized, controlled study with 262 and 87 participants with T2D were enrolled in the CCI and usual care (UC) groups, respectively. RESULTS: Significant changes from baseline to 2 years in the CCI group included: HbA1c (-12% from 7.7+0.1%); fasting glucose (-18% from 163.67+3.90 mg/dL); fasting insulin (-42% from 27.73+1.26 pmol L-1); weight (-10% from 114.56+0.60 kg); systolic blood pressure (-4% from 131.7+0.9 mmHg); diastolic blood pressure (-4% from 81.8+0.5 mmHg); triglycerides (-22% from 197.2+9.1 mg/dL); HDL-C (+19% from 41.8+0.9 mg/dL), and liver alanine transaminase (-21% from 29.16+0.97 U/L). Spine bone mineral density in the CCI group was unchanged. Glycemic control medication use (excluding metformin) among CCI participants declined (from 56.9% to 26.8%, P=1.3x10-11) including prescribed insulin (-62%) and sulfonylureas (-100%). The UC group had no significant changes in these parameters (except uric acid and anion gap) or diabetes medication use. There was also significant resolution of diabetes (reversal, 53.5%; remission, 17.6%) in the CCI group but not in UC. All the reported improvements had p-values <0.00012. CONCLUSIONS: The CCI sustained long-term beneficial effects on multiple clinical markers of diabetes and cardiometabolic health at 2 years while utilizing less medication. The intervention was also effective in the resolution of diabetes and visceral obesity, with no adverse effect on bone health. Clinical trial registration ID #NCT02519309

313: Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics
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Posted to bioRxiv 05 Jun 2019

Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics
309 downloads biophysics

Kyle W. East, Jocelyn C. Newton, Uriel N. Morzan, Atanu Acharya, Erin Skeens, Gerwald Jogl, Victor S. Batista, Giulia Palermo, George P. Lisi

CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and Gaussian accelerated Molecular Dynamics (GaMD) simulations - flanked by mixed machine learning and structure-based prediction of NMR chemical shifts - are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond timescale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in the full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome editing capability

314: Unraveling the polygenic architecture of complex traits using blood eQTL meta-analysis
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Posted to bioRxiv 19 Oct 2018

Unraveling the polygenic architecture of complex traits using blood eQTL meta-analysis
308 downloads genomics

Urmo Võsa, Annique Claringbould, Harm-Jan Westra, Marc Jan Bonder, Patrick Deelen, Biao Zeng, Holger Kirsten, Ashis Saha, Roman Kreuzhuber, Silva Kasela, Natalia Pervjakova, Isabel Alvaes, Marie-Julie Fave, Mawusse Agbessi, Mark Christiansen, Rick Jansen, Ilkka Seppälä, Lin Tong, Alexander Teumer, Katharina Schramm, Gibran Hemani, Joost Verlouw, Hanieh Yaghootkar, Reyhan Sönmez, Andrew A. Andrew, Viktorija Kukushkina, Anette Kalnapenkis, Sina Rüeger, Eleonora Porcu, Jaanika Kronberg-Guzman, Johannes Kettunen, Joseph Powell, Bernett Lee, Futao Zhang, Wibowo Arindrarto, Frank Beutner, BIOS Consortium, Harm Brugge, i2QTL Consortium, Julia Dmitrieva, Mahmoud Elansary, Benjamin P. Fairfax, Michel Georges, Bastiaan T. Heijmans, Mika Kähönen, Yungil Kim, Julian C Knight, Peter Kovacs, Knut Krohn, Shuang Li, Markus Loeffler, Urko M Marigorta, Hailang Mei, Yukihide Momozawa, Martina Müller-Nurasyid, Matthias Nauck, Michel Nivard, Brenda Penninx, Jonathan Pritchard, Olli Raitakari, Olaf Rotzschke, Eline P Slagboom, Coen D.A. Stehouwer, Michael Stumvoll, Patrick Sullivan, Peter A.C. 't Hoen, Joachim Thiery, Anke Tönjes, Jenny van Dongen, Maarten van Iterson, Jan Veldink, Uwe Völker, Cisca Wijmenga, Morris Swertz, Anand Andiappan, Grant W. Montgomery, Samuli Ripatti, Markus Perola, Zoltan Kutalik, Emmanouil Dermitzakis, Sven Bergmann, Timothy Frayling, Joyce van Meurs, Holger Prokisch, Habibul Ahsan, Brandon Pierce, Terho Lehtimäki, Dorret Boomsma, Bruce M Psaty, Sina A. Gharib, Philip Awadalla, Lili Milani, Willem H. Ouwehand, Kate Downes, Oliver Stegle, Alexis Battle, Jian Yang, Peter M. Visscher, Markus Scholz, Gregory Gibson, Tõnu Esko, Lude Franke

While many disease-associated variants have been identified through genome-wide association studies, their downstream molecular consequences remain unclear. To identify these effects, we performed cis- and trans-expression quantitative trait locus (eQTL) analysis in blood from 31,684 individuals through the eQTLGen Consortium. We observed that cis-eQTLs can be detected for 88% of the studied genes, but that they have a different genetic architecture compared to disease-associated variants, limiting our ability to use cis-eQTLs to pinpoint causal genes within susceptibility loci. In contrast, trans-eQTLs (detected for 37% of 10,317 studied trait-associated variants) were more informative. Multiple unlinked variants, associated to the same complex trait, often converged on trans-genes that are known to play central roles in disease etiology. We observed the same when ascertaining the effect of polygenic scores calculated for 1,263 genome-wide association study (GWAS) traits. Expression levels of 13% of the studied genes correlated with polygenic scores, and many resulting genes are known to drive these traits.

315: The evolutionary history of 2,658 cancers
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Posted to bioRxiv 11 Jul 2017

The evolutionary history of 2,658 cancers
307 downloads cancer biology

Moritz Gerstung, Clemency Jolly, Ignaty Leshchiner, Stefan C. Dentro, Santiago Gonzalez Rosado, Daniel Rosebrock, Thomas J. Mitchell, Yulia Rubanova, Pavana Anur, Kaixan Yu, Maxime Tarabichi, Amit Deshwar, Jeff Wintersinger, Kortine Kleinheinz, Ignacio Vazquez-Garcia, Kerstin Haase, Lara Jerman, Subhajit Sengupta, Geoff Macintyre, Salem Malikic, Nilgun Donmez, Dimitri G. Livitz, Marek Cmero, Jonas Demeulemeester, Steve Schumacher, Yu Fan, Xiaotong Yao, Juhee Lee, Matthias Schlesner, Paul C. Boutros, David D. Bowtell, Hongtu Zhu, Gad Getz, Marcin Imielinski, Rameen Beroukhim, S. Cenk Sahinalp, Yuan Ji, Martin Peifer, Florian Markowetz, Ville Mustonen, Ke Yuan, Wenyi Wang, Quaid D Morris, Paul T. Spellman, David C. Wedge, Peter Van Loo, PCAWG Evolution and Heterogeneity Working Group, PCAWG network

Cancer develops through a process of somatic evolution. Here, we reconstruct the evolutionary history of 2,778 tumour samples from 2,658 donors spanning 39 cancer types. Characteristic copy number gains, such as trisomy 7 in glioblastoma or isochromosome 17q in medulloblastoma, are found amongst the earliest events in tumour evolution. The early phases of oncogenesis are driven by point mutations in a restricted set of cancer genes, often including biallelic inactivation of tumour suppressors. By contrast, increased genomic instability, a more than three-fold diversification of driver genes, and an acceleration of mutational processes are features of later stages. Clock-like mutations yield estimates for whole genome duplications and subclonal diversification in chronological time. Our results suggest that driver mutations often precede diagnosis by many years, and in some cases decades. Taken together, these data reveal common and divergent trajectories of cancer evolution, pivotal for understanding tumour biology and guiding early cancer detection.

316: Deep Learning Interpretation of Echocardiograms
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Posted to bioRxiv 24 Jun 2019

Deep Learning Interpretation of Echocardiograms
307 downloads bioengineering

Amirata Ghorbani, David Ouyang, Abubakar Abid, Bryan He, Jonathan H. Chen, Robert A. Harrington, David H. Liang, Euan A. Ashley, James Y. Zou

Echocardiography uses ultrasound technology to capture high temporal and spatial resolution images of the heart and surrounding structures and is the most common imaging modality in cardiovascular medicine. Using convolutional neural networks on a large new dataset, we show that deep learning applied to echocardiography can identify local cardiac structures, estimate cardiac function, and predict systemic phenotypes that modify cardiovascular risk but not readily identifiable to human interpretation. Our deep learning model, EchoNet, accurately identified the presence of pacemaker leads (AUC = 0.89), enlarged left atrium (AUC = 0.85), normal left ventricular wall thickness (AUC = 0.75), left ventricular end systolic and diastolic volumes(R^2 = 0.73 and R^2 = 0.68), and ejection fraction (R^2 = 0.48) as well as predicted systemic phenotypes of age (R^2 = 0.46), sex (AUC = 0.88), weight (R^2 = 0.56), and height (R^2 = 0.33). Interpretation analysis validates that EchoNet shows appropriate attention to key cardiac structures when performing human-explainable tasks and highlight hypothesis-generating regions of interest when predicting systemic phenotypes difficult for human interpretation. Machine learning on echocardiography images can streamline repetitive tasks in the clinical workflow, standardize interpretation in areas with insufficient qualified cardiologists, and more consistently produce echocardiographic measurements.

317: Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology
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Posted to bioRxiv 02 Jul 2019

Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology
306 downloads immunology

Travis K Hughes, Marc H Wadsworth, Todd M Gierahn, Tran Do, David Weiss, Priscilla R. Andrade, Feiyang Ma, Bruno J. de Andrade Silva, Shuai Shao, Lam C Tsoi, Jose Ordovas-Montanes, Johann E Gudjonsson, Robert L Modlin, J Christopher Love, Alex K Shalek

The development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies has empowered the characterization of complex biological samples by dramatically increasing the number of constituent cells that can be examined concurrently. Nevertheless, these approaches typically recover substantially less information per-cell as compared to lower-throughput microtiter plate-based strategies. To uncover critical phenotypic differences among cells and effectively link scRNA-Seq observations to legacy datasets, reliable detection of phenotype-defining transcripts – such as transcription factors, affinity receptors, and signaling molecules – by these methods is essential. Here, we describe a substantially improved massively-parallel scRNA-Seq protocol we term Seq-Well S^3 (“Second-Strand Synthesis”) that increases the efficiency of transcript capture and gene detection by up to 10- and 5-fold, respectively, relative to previous iterations, surpassing best-in-class commercial analogs. We first characterized the performance of Seq-Well S^3 in cell lines and PBMCs, and then examined five different inflammatory skin diseases, illustrative of distinct types of inflammation, to explore the breadth of potential immune and parenchymal cell states. Our work presents an essential methodological advance as well as a valuable resource for studying the cellular and molecular features that inform human skin inflammation.

318: H3K9me2 orchestrates inheritance of spatial positioning of peripheral heterochromatin through mitosis
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Posted to bioRxiv 21 Jun 2019

H3K9me2 orchestrates inheritance of spatial positioning of peripheral heterochromatin through mitosis
306 downloads cell biology

Andrey Poleshko, Cheryl L Smith, Son C. Nguyen, Priya Sivaramakrishnan, John Isaac Murray, Melike Lakadamyali, Eric F. Joyce, Raj Jain, Jonathan A Epstein

Cell-type-specific 3D organization of the genome is unrecognizable during mitosis. It remains unclear how essential positional information is transmitted through cell division such that a daughter cell recapitulates the spatial genome organization of the parent. Lamina-associated domains (LADs) are regions of repressive heterochromatin positioned at the nuclear periphery that vary by cell type and contribute to cell-specific gene expression. Here we show that histone 3 lysine 9 dimethylation (H3K9me2) specifically marks peripheral heterochromatin and is retained through mitosis when phosphorylation of histone 3 serine 10 shields the H3K9me2 mark allowing for dissociation from the nuclear lamina. The H3K9me2 modification of peripheral heterochromatin ensures that positional information is safeguarded through cell division such that individual LADs are re-established at the nuclear periphery in daughter nuclei. Thus, H3K9me2 acts as a 3D architectural mitotic guidepost. Our data establish a mechanism for epigenetic memory and inheritance of spatial organization of the genome.

319: Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting
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Posted to bioRxiv 06 Jun 2019

Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting
305 downloads biochemistry

Glenn Cremers, Bas Rosier, Roger Riera Brillas, Lorenzo Albertazzi, Tom de Greef

The combination of the specificity of antibodies and the programmability of DNA nanotechnology has provid-ed the scientific community with a powerful tool to label and unambiguously distinguish a large number of subcellular targets using fluorescence-based read-out methods. While primary antibodies are commercially available for a large class of targets, a general stoichiometric site-specific DNA labelling strategy for this affinity reagent is lacking. Here, we present a universal, site-selective, conjugation method using a small photocross-linkable protein G adaptor that allows labelling of antibodies of different host species with a controlled number of short oligonucleotides (ODNs). Importantly, we illustrate that this conjugation method can be directly performed on commercially-available primary antibodies, on a small scale and without cross-reactivity towards other proteins, such as bovine serum albumin. In addition, we present a general, benchtop-compatible strategy to purify DNA-labeled antibodies without loss of function. The application of protein G-ODN labelled primary antibodies is demonstrated by employing three well-known methods for detecting subcellular targets using fluorescent read-out, including flow cytometry, DNA-PAINT, and dSTORM. This work thus establishes a general and efficient platform for the synthesis of a library of unique ODN-antibody conjugates, facilitating the broader use of DNA-based programmable tags for multiplexed labelling to identify subcellular features with nanometer-precision, improving our understanding of cellular structure and function.

320: Modified TCA/acetone precipitation of plant proteins for proteomic analysis
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Posted to bioRxiv 01 Aug 2018

Modified TCA/acetone precipitation of plant proteins for proteomic analysis
305 downloads biochemistry

Liangjie Niu, Hang Zhang, Zhaokun Wu, Wei Wang, Hui Liu, Xiaolin Wu

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

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