bluuebunny/crossref_metadata_2025 huggingface dataset:
A pancreas specificPtf1a-driven Cre mouse line causes paternally transmitted germline recombination
Authors: Kabacaoglu Derya , Lesina Marina , Algül Hana
Published: March 2020

Cre-loxP recombination system is a commonly used tool to achieve site-specific genetic manipulation in genome. For multiple Cre driver mouse lines, parental transmissions of recombined flox alleles are reported.Ptf1a-driven Cre lines are widely used to achieve genetic manipulation in a pancreas specific manner. Herein, we report germline recombination in breedings whenCreallele is retained paternally inPtf1atm1(cre)Hnak. The germline recombination frequency changed depending on the target allele. Therefore, unless the reporter allele is on the target gene, the reporter activity is to be validated. Overall, we highlight that allPtf1a-driven Cre mouse lines should be genotyped for possible germline recombination and we advise the maternal transmission of theCreto progeny.
Leptin Receptors in RIP-Cre25Mgnneurons Mediate Anti-Dyslipidemia Effects of Leptin in Insulin-Deficient Male Mice
Authors: Singha Ashish , Palavicini Juan Pablo , Pan Meixia , Sandoval Darleen , Han Xianlin , Fujikawa Teppei
Published: March 2020

Leptin is a potent endocrine hormone produced by adipose tissue and regulates a broad range of metabolism including glucose and lipid metabolism, with and without insulin. It is evident that central leptin signaling can lower hyperglycemia in insulin-deficient rodents via multiple mechanisms including restoration of dyslipidemia. However, the specific neurons that regulate these glucose-lowering and anti-dyslipidemia effects of leptin remain unidentified. Here we report that leptin receptors (LEPRs) in neurons expressing Cre recombinase driven by a short fragment of a promoter region ofIns2gene (RIP-Cre25Mgnneurons) are required for central leptin signaling to reverse hyperglycemia and dyslipidemia in insulin-deficient mice. Ablation of LEPRs in RIP-Cre25Mgnneurons completely blocks glucose-lowering effects of leptin in insulin-deficient mice. Further investigations reveal that insulin-deficient mice lacking LEPRs in RIP-Cre25Mgnneurons (RIP-CreΔLEPRmice) exhibit greater lipid levels in blood and liver compared to wild-type controls, and that leptin injection into the brain does not suppress dyslipidemia in insulin-deficient RIP-CreΔLEPRmice. Leptin administration into the brain combined with acipimox, which lowers blood lipids by suppressing triglyceride lipase activity, can restore normal glycemia in insulin-deficient RIP-CreΔLEPRmice, suggesting that excess circulating lipids are a driving-force of hyperglycemia in insulin-deficient RIP-CreΔLEPRmice. Collectively, our data demonstrate that LEPRs in RIP-Cre25Mgnneurons significantly contribute to glucose-lowering effects of leptin in an insulin-independent manner by suppression of dyslipidemia.
Genome-wide Screens Identify Lineage- and Tumor Specific-Genes Modulating MHC-I and MHC-II Immunosurveillance in Human Lymphomas
Authors: Dersh Devin , Phelan James D. , Gumina Megan E. , Wang Boya , Arbuckle Jesse H. , Holly Jaroslav , Kishton Rigel J. , Markowitz Tovah E. , Seedhom Mina O. , Fridlyand Nathan , Wright George W. , Huang Da Wei , Ceribelli Michele , Thomas Craig J. , Lack Justin B. , Restifo Nicholas P. , Kristie Thomas M. , Staudt Louis M. , Yewdell Jonathan W.
Published: March 2020

Tumors frequently subvert MHC class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance. To better define the regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of novel genes that positively and negatively modulate MHC-I cell surface levels. Identified genes cluster in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Many genes exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors display genetic alterations in multiple regulators. We establish that the HSP90 co-chaperone SUGT1 is a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhances DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.
Character evolution of modern fly-speck fungi and implications for interpreting thyriothecial fossils
Authors: Renard Ludovic Le , Firmino André L. , Pereira Olinto L. , Stockey Ruth A. , Berbee Mary. L.
Published: March 2020

PREMISE OF THE STUDY

Fossils show that fly-speck fungi have been reproducing with small, black thyriothecia on leaf surfaces for ∼250 million years. We analyze morphological characters of extant thyriothecial fungi to develop a phylogenetic framework for interpreting fossil taxa.

METHODS

We placed 59 extant fly-speck fungi in a phylogeny of 320 Ascomycota using nuclear ribosomal large and small subunit sequences, including newly determined sequences from nine taxa. We reconstructed ancestral character states using BayesTraits and maximum likelihood after coding 11 morphological characters based on original observations and literature. We analyzed the relationships of three previously published Mesozoic fossils using parsimony and our morphological character matrix, constrained by the molecular phylogeny.

KEY RESULTS

Thyriothecia evolved convergently in multiple lineages of superficial, leaf- inhabiting ascomycetes. The radiate and ostiolate scutellum organization is restricted to Dothideomycetes. Scutellum initiation by intercalary septation of a single hypha characterizes Asterinales and Asterotexiales, and initiation by coordinated growth of two or more adjacent hyphae characterizes Aulographaceae (orderincertae sedis). Scutella in Microthyriales are initiated apically on a lateral hyphal branch. Patterns of hyphal branching in scutella contribute to distinguishing among orders. Parsimony resolves three fossil taxa as Dothideomycetes; one is further resolved as a member of a Microthyriales-Zeloasperisporiales clade within Dothideomycetes.

CONCLUSIONS

This is the most comprehensive systematic study of thyriothecial fungi and their relatives to date. Parsimony analysis of the matrix of character states of modern taxa provides an objective basis for interpreting fossils, leading to insights into morphological evolution and geological ages of Dothideomycetes clades.
Light microscopy of proteins in their ultrastructural context
Authors: M’Saad Ons , Bewersdorf Joerg
Published: March 2020

Resolving the distribution of specific proteins at the nanoscale in the structural context of the cell is a major challenge in fluorescence microscopy. Here we present a new concept that decrowds the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins. This combination makes labeling of the proteome efficient enough that local protein densities are revealed and the cellular nanoarchitecture can be visualized by standard light microscopy.
Discovery of the world’s highest-dwelling mammal
Authors: Storz Jay F. , Quiroga-Carmona Marcial , Opazo Juan C. , Bowen Thomas , Farson Matthew , Steppan Scott J. , D’Elía Guillermo
Published: March 2020

Environmental limits of animal life are invariably revised upwards when the animals themselves are investigated in their natural habitats. Here we report results of a scientific mountaineering expedition to survey the high-altitude rodent fauna of Volcán Llullaillaco in the Puna de Atacama of northern Chile, an effort motivated by video documentation of mice (genusPhyllotis) at a record altitude of 6205 m. Among numerous trapping records at altitudes >5000 m, we captured a specimen of the yellow-rumped leaf-eared mouse (Phyllotis xanthopygus rupestris) on the very summit of Llullaillaco at 6739 m. This summit specimen represents an altitudinal world record for mammals, far surpassing all specimen-based records from the Himalayas and elsewhere in the Andes. This discovery suggests that we may have generally underestimated the altitudinal range limits and physiological tolerances of small mammals simply because the world’s highest summits remain relatively unexplored by biologists.
Modeling Regulatory Network Topology Improves Genome-Wide Analyses of Complex Human Traits
Authors: Zhu Xiang , Duren Zhana , Wong Wing Hung
Published: March 2020

Genome-wide association studies (GWAS) have cataloged many significant associations between genetic variants and complex traits. However, most of these findings have unclear biological significance, because they often have small effects and occur in non-coding regions. Integration of GWAS with gene regulatory networks addresses both issues by aggregating weak genetic signals within regulatory programs. Here we develop a Bayesian framework that integrates GWAS summary statistics with regulatory networks to infer genetic enrichments and associations simultaneously. Our method improves upon existing approaches by explicitly modeling network topology to assess enrichments, and by automatically leveraging enrichments to identify associations. Applying this method to 18 human traits and 38 regulatory networks shows that genetic signals of complex traits are often enriched in interconnections specific to trait-relevant cell types or tissues. Prioritizing variants within enriched networks identifies known and new trait-associated genes revealing novel biological and therapeutic insights.
Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in Rhesus macaques
Authors: Deng Wei , Bao Linlin , Gao Hong , Xiang Zhiguang , Qu Yajin , Song Zhiqi , Gong Shunran , Liu Jiayi , Liu Jiangning , Yu Pin , Qi Feifei , Xu Yanfeng , Li Fengli , Xiao Chong , Lv Qi , Xue Jing , Wei Qiang , Liu Mingya , Wang Guanpeng , Wang Shunyi , Yu Haisheng , Liu Xing , Zhao Wenjie , Han Yunlin , Qin Chuan
Published: March 2020

The outbreak of Corona Virus Disease 2019 caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) is highly transmitted. The potential extra-respiratory transmission routes remain uncertain. Five rhesus macaques were inoculated with 1×106TCID50of SARS-CoV-2 via conjunctival (CJ), intratracheal (IT), and intragastric (IG) routes, respectively. Remarkably, the CJ inoculated-macaques developed mild interstitial pneumonia and viral load was detectable in the conjunctival swabs at 1 days post-inoculation (dpi). Only via IT inoculation, viral load was detected in the anal swab at 1-7 dpi and macaque showed weight loss. However, viral load was undetectable after IG inoculation. Comparatively, viral load was higher in the nasolacrimal system but lesions of lung were relatively mild and local via CJ inoculation compared with that via IT inoculation, demonstrating distinct characteristics of virus dispersion. Both the two routes affected the alimentary tract. Therefore the clinicians need to protect eye while working with patients.
FITNESS BENEFITS TO BACTERIA OF CARRYING PROPHAGES AND PROPHAGE-ENCODED ANTIBIOTIC-RESISTANCE GENES PEAK IN DIFFERENT ENVIRONMENTS
Authors: Wendling Carolin C. , Refardt Dominik , Hall Alex R.
Published: March 2020

Bacteria can acquire antibiotic resistance genes (ARGs) via prophages, phage genomes integrated into bacterial chromosomes. Such prophages may influence bacterial fitness via increased antibiotic resistance, protection from further phage infection, or by switching to a lytic lifecycle that releases free phages which can infect phage-susceptible competitors. We expect these effects to depend on environmental conditions because of, for example, environment-dependent induction of the lytic lifecycle. However, our understanding of how costs and benefits of prophage-encoded ARGs vary across environments remains limited. Here, by studying prophages with and without ARGs inEscherichia coli, we distinguished between effects of prophages alone and ARGs they carry. In competition with prophage-free strains, fitness benefits from prophages and ARGs peaked in different environments. Prophage carriage was most beneficial in conditions where induction of the lytic lifecycle was common, whereas ARGs were more beneficial in the presence of antibiotics and when prophage induction was lower. Acquisition of prophage-encoded ARGs by competing phage-susceptible strains was most common when prophage induction, and therefore the amount of free phages, was high. Thus, selection on prophages and ARGs varies independently across environments, which is important for predicting the spread of mobile/integrating genetic elements and their role in antibiotic resistance evolution.
Targeted genome editing inNicotiana tabacumusing inducible CRISPR/Cas9 system
Authors: Ren Chong , Liu Yanfei , Wang Xida , Guo Yuchen , Fan Peige , Li Shaohua , Liang Zhenchang
Published: March 2020

Targeted genome editing has been achieved in multiple plant species using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, in which theCas9gene is usually driven by constitutive promoters. However, constitutive expression ofCas9is not necessary and can be harmful to plant development. In this study, we developed an estrogen-inducible CRISPR/Cas9 system by taking advantage of the chimeric transcription activator XVE and tested the efficacy of this inducible system inNicotiana tabacumby targeting the phytoene desaturase (NtPDS) gene, whose mutation resulted in albino phenotypes. Treatment of four independent transgenic lines with exogenous estradiol successfully induced targeted mutagenesis inNtPDS. Sanger sequencing assay uncovered the presence of indel mutations (nucleotides insertions or deletions) at the target site as expected, and at least two types of mutations were identified for each line. Transgenic plants with mutatedNtPDSgene after estradiol treatment exhibited pale green or incomplete albino leaves. Moreover, the expression ofCas9in transgenic plants was strongly induced by estradiol treatment. Our results demonstrate the efficacy of XVE-based CRISPR/Cas9 system inN. tabacum, and the system reported here promises to be a useful approach for conditional genome editing, which would facilitate the study of genes of interest, especially those developmentally important genes.
Tales of 1,008 Small Molecules: Phenomic Profiling through Live-cell Imaging in a Panel of Reporter Cell Lines
Authors: Cox Michael J. , Jaensch Steffen , Van de Waeter Jelle , Cougnaud Laure , Seynaeve Daan , Benalla Soulaiman , Koo Seong Joo , Van Den Wyngaert Ilse , Neefs Jean-Marc , Malkov Dmitry , Bittremieux Mart , Steemans Margino , Peeters Pieter J. , Wegner Jörg Kurt , Ceulemans Hugo , Gustin Emmanuel , Chong Yolanda T. , Göhlmann Hinrich W.H.
Published: March 2020

Phenomic profiles are high-dimensional sets of readouts that can comprehensively capture the biological impact of chemical and genetic perturbations in cellular assay systems. Phenomic profiling of compound libraries can be used for compound target identification or mechanism of action (MoA) prediction and other applications in drug discovery. To devise an economical set of phenomic profiling assays, we assembled a library of 1,008 approved drugs and well-characterized tool compounds manually annotated to 218 unique MoAs, and we profiled each compound at four concentrations in live-cell, high-content imaging screens against a panel of 15 reporter cell lines, which expressed a diverse set of fluorescent organelle and pathway markers in three distinct cell lineages. For 41 of 83 testable MoAs, phenomic profiles accurately ranked the reference compounds (AUC-ROC ≥0.9). MoAs could be better resolved by screening compounds at multiple concentrations than by including replicates at a single concentration. Screening additional cell lineages and fluorescent markers increased the number of distinguishable MoAs but this effect quickly plateaued. There remains a substantial number of MoAs that were hard to distinguish from others under the current study’s conditions. We discuss ways to close this gap, which will inform the design of future phenomic profiling efforts.
Architecture and assembly dynamics of the essential mitochondrial TIM chaperone systems
Authors: Weinhäupl Katharina , Wang Yong , Hessel Audrey , Brennich Martha , Lindorff-Larsen Kresten , Schanda Paul
Published: March 2020

The mitochondrial Tim chaperones are responsible for the transport of membrane proteins across the inter-membrane space to the inner and outer mitochondrial membranes. TIM9·10, a hexameric 70 kDa protein complex formed by 3 copies of Tim9 and Tim10, guides its clients across the aqueous compartment. The TIM9·10·12 complex is the anchor point at the inner-membrane insertase complex TIM22. The subunit composition of the TIM9·10·12 complex remains debated. Joint NMR, small-angle X-ray scattering and MD simulation data allow us to derive a structural model of the TIM9·10·12 assembly, which has a 2:3:1 stoichiometry (Tim9:Tim10:Tim12). We find that both TIM9·10 and TIM9·10·12 hexamers are in a dynamic equilibrium with their constituent subunits, exchanging on a minutes time scale. Residue-resolved NMR data establish that the subunits exhibit large conformational dynamics: when the conserved cysteines of the CX3C-Xn-CX3C motifs are formed, short marginally stableα-helices are formed, and these are fully stabilized only upon formation of the mature hexameric chaperone. We propose that the continuous subunit exchange is a means of mitochondria to control their level of inter-membrane space chaperones, and thus rapidly adapt to the cellular state.
Detection of M-Protein in Acetonitrile Precipitates of Serum using MALDI-TOF Mass Spectrometry
Authors: Mehra Nikita , Gopisetty Gopal , Jayavelu S , Arivazhagan Rajamanickam , Sundersingh Shirley , Karunakaran Parathan , Kalaiyarasi Jayachandran Perumal , Kannan Krishnarathinam , Radhakrishnan Venkatraman , Sagar Tenali Gnana , Rajkumar Thangarajan
Published: March 2020

Purpose of the research

Multiple myeloma and plasmacytomas belong to a group of disorders, namely plasma cell dyscrasias and are identified by the presence of a monoclonal protein (M-protein). MALDI-TOF-mass spectrometry (MS) has demonstrated superior analytical sensitivity for the detection of M-protein and is now used for screening of M-protein at some centres. We present the results of an alternative methodology for M-protein analysis by MALDI-TOF MS.

Methods

Serum samples from patients with newly diagnosed multiple myeloma or plasmacytoma with positive M-protein detected by serum protein electrophoresis, immunofixation electrophoresis and serum free light chain analysis, underwent direct reagent-based extraction process using Acetonitrile (ACN) precipitation. Serumκandλlight chains were validated using immunoenrichment by anti-κand anti-λbiotin-labelled antibodies immobilised on streptavidin magnetic beads. MALDI-TOF MS measurements were obtained for intact proteins using alpha-cyano-4-hydroxycinnamic acid as matrix. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein.

Principle results

Characteristic M-protein peaks were observed in the ACN precipitates of serum in the predicted mass ranges forκandλ. Theκandλpeaks were confirmed by immunoenrichment analysis. Twenty-seven patient samples with either newly diagnosed multiple myeloma or plasmacytoma with monoclonal gammopathy detected by the standard methods were chosen for Acetonitrile precipitation and analysed by MALDI-TOF MS. All 27 patient samples demonstrated a peak suggestive of M-protein with mass/charge(m/z)falling within theκandλrange. The concordance rate with serum immunofixation electrophoresis and free light chain analysis was above 90%.

Major conclusions

We report the results of a low-cost reagent-based extraction process using Acetonitrile precipitation to enrich forκandλlight chains, which can be used for the screening and qualitative analysis of M-protein.
The COMPASS-like complex modulates fungal development and pathogenesis by regulating H3K4me3-mediated targeted gene expression inMagnaporthe oryzae
Authors: Zhou Sida , Sun Wanyu , Zhao Xinyu , Xu Yang , Zhang Mengyu , Yin Yue , Pan Song , He Dan , Shen Mi , Yang Jun , Liu Xiuying , Zheng Qi , Wang Weixiang
Published: March 2020

Histone H3K4 methylation is catalysed by the multi-protein complex known as the Set1/COMPASS or MLL/COMPASS-like complex, an element that is highly evolutionarily conserved from yeast to humans. However, the components and mechanisms by which the COMPASS-like complex targets the H3K4 methylation of plant pathogenic genes in fungi remain elusive. Here we present a comprehensive analysis combining biochemical, molecular, and genome-wide approaches to characterize the roles of the COMPASS-like family inMagnaporthe oryzae, a model plant fungal pathogen. We purified and identified six conserved subunits of COMPASS from the rice blast fungusM. oryzae, i.e., MoBre2 (Cps60/ASH2L), MoSpp1 (Cps40/Cfp1), MoSwd2 (Cps35), MoSdc1 (Cps25/DPY30), MoSet1 (MLL/ALL) and MoRbBP5 (Cps50), using an affinity tag on MoBre2. We determined the SPRY domain of MoBre2 can recognize directly with DPY30 domain of MoSdc1in vitro.Furthermore, we found that deletion of the genes encoding COMPASS subunits of MoBre2, MoSpp1 and MoSwd2 caused similar defects regarding invasive hyphal development and pathogenicity. Genome-wide profiling of H3K4me3 revealed that the it has remarkable co-occupancy at the TSS regions of target genes. Significantly, these target genes are often involved in spore germination and pathogenesis. Decreased gene expression caused by the deletion ofMoBre2,MoSwd2orMoSpp1gene was highly correlated with decrease in H3K4me3. Taken together, these results suggest that MoBre2, MoSpp1, and MoSwd2 function as a whole COMPASS complex, contributing to fungal development and pathogenesis by regulating H3K4me3-targeted genes inM. oryzae.
Lack of Reinfection in Rhesus Macaques Infected with SARS-CoV-2
Authors: Bao Linlin , Deng Wei , Gao Hong , Xiao Chong , Liu Jiayi , Xue Jing , Lv Qi , Liu Jiangning , Yu Pin , Xu Yanfeng , Qi Feifei , Qu Yajin , Li Fengdi , Xiang Zhiguang , Yu Haisheng , Gong Shuran , Liu Mingya , Wang Guanpeng , Wang Shunyi , Song Zhiqi , Liu Ying , Zhao Wenjie , Han Yunlin , Zhao Linna , Liu Xing , Wei Qiang , Qin Chuan
Published: March 2020

A global pandemic of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is ongoing spread. It remains unclear whether the convalescing patients have a risk of reinfection. Rhesus macaques were rechallenged with SARS-CoV-2 during an early recovery phase from initial infection characterized by weight loss, interstitial pneumonia and systemic viral dissemination mainly in respiratory and gastrointestinal tracts. The monkeys rechallenged with the identical SARS-CoV-2 strain have failed to produce detectable viral dissemination, clinical manifestations and histopathological changes. A notably enhanced neutralizing antibody response might contribute the protection of rhesus macaques from the reinfection by SARS-CoV-2. Our results indicated that primary SARS-CoV-2 infection protects from subsequent reinfection.

One Sentence Summary

Neutralizing antibodies against SARS-CoV-2 might protect rhesus macaques which have undergone an initial infection from reinfection during early recovery days.
Classification and Specific Primer Design for Accurate Detection of SARS-CoV-2 Using Deep Learning
Authors: Lopez-Rincon Alejandro , Tonda Alberto , Mendoza-Maldonado Lucero , Mulders Daphne G.J.C. , Molenkamp Richard , Perez-Romero Carmina A. , Claassen Eric , Garssen Johan , Kraneveld Aletta D.
Published: March 2020

In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from available repositories, separating the genome of different virus strains from the Coronavirus family with considerable accuracy. The network’s behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are first validated on samples from other repositories, and proven able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets on existing datasets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n=6 previously tested positive), delivering a sensibility similar to routine diagnostic methods, and 100% specificity. In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from NGDC, separating the genome of different virus strains from the Coronavirus family with accuracy 98.73%. The network’s behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are validated on samples from NCBI and GISAID, and proven able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n=6 previously tested positive), delivering a sensibility similar to routine diagnostic methods, and 100% specificity. The proposed methodology has a substantial added value over existing methods, as it is able to both identify promising primer sets for a virus from a limited amount of data, and deliver effective results in a minimal amount of time. Considering the possibility of future pandemics, these characteristics are invaluable to promptly create specific detection methods for diagnostics.
Differential Antibody Recognition by Novel SARS-CoV-2 and SARS-CoV Spike Protein Receptor Binding Domains: Mechanistic Insights and Implications for the Design of Diagnostics and Therapeutics
Authors: D’Annessa Ilda , Marchetti Filippo , Colombo Giorgio
Published: March 2020

The appearance of the novel betacoronavirus SARS-CoV-2 represents a major threat to human health, and its diffusion around the world is causing dramatic consequences. The knowledge of the 3D structures of SARS-CoV-2 proteins can facilitate the development of therapeutic and diagnostic molecules. Specifically, comparative analyses of the structures of SARS-CoV-2 proteins and homologous proteins from previously characterized viruses, such as SARS-CoV, can reveal the common and/or distinctive traits that underlie the mechanisms of recognition of cell receptors and of molecules of the immune system.

Herein, we apply our recently developed energy-based methods for the prediction of antibody-binding epitopes and protein-protein interaction regions to the Receptor Binding Domain (RBD) of the Spike proteins from SARS-CoV-2 and SARS-CoV. Our analysis focusses only on the study of the structure of RBDs in isolation, without making use of any previous knowledge of binding properties. Importantly, our results highlight structural and sequence differences among the regions that are predicted to be immunoreactive and bind/elicit antibodies. These results provide a rational basis to the observation that several SARS-CoV RDB-specific monoclonal antibodies fail to appreciably bind the SARS-CoV-2 counterpart. Furthermore, we correctly identify the region of SARS-CoV-2 RBD that is engaged by the cell receptor ACE2 during viral entry into host cells.

The data, sequences and structures we present here can be useful for the development of novel therapeutic and diagnostic interventions.
Flexibility and constraint in preimplantation gene regulation in mouse
Authors: Conine Colin C. , Krykbaeva Marina , Song Lina , Brewster Robert C. , Friedman Nir , Rando Oliver J.
Published: March 2020

Although many features of embryonic development exhibit remarkable stability in the face of environmental perturbations, it is also clear that some aspects of early embryogenesis can be modulated by non-genetic influences during and after fertilization. Among potential perturbations experienced during reproduction, understanding the consequences of differing ex vivo fertilization methods at a molecular level is imperative for comprehending both the basic biology of early development and the potential consequences of assisted reproduction. Here, we set out to explore stable and flexible aspects of preimplantation gene expression using single-embryo RNA-sequencing of mouse embryos fertilized by natural mating, in vitro fertilization, or intracytoplasmic sperm injection, as well as oocytes parthenogenetically activated to develop (parthenotes). This dataset comprises a resource of over eight hundred individual embryos, which we use for three primary analyses. First, we characterize the effects of each fertilization method on early embryonic gene regulation, most notably finding decreased expression of trophectoderm markers at later stages of preimplantation development in ICSI embryos. Second, we find massive gene misregulation in parthenotes beyond the expected defects in imprinted gene expression, and show that many of these changes can be suppressed by sperm total RNA. Finally, we make use of the single-embryo resolution of our dataset to identify both stably-expressed genes and highly-variable genes in the early mouse embryo. Together, our data provide a detailed survey of the molecular consequences of different fertilization methods, establish parthenotes as a “tabula rasa” for understanding the role for sperm RNAs in preimplantation gene regulation, and identify subtypes of preimplantation embryos based on their expression of epivariable gene modules.
Parallel social information processing circuits are differentially impacted in autism
Authors: Lewis Eastman M. , Stein-O’Brien Genevieve L. , Patino Alejandra V. , Nardou Romain , Grossman Cooper D. , Brown Matthew , Bangamwabo Bidii , Ndiaye Ndeye , Giovinazzo Daniel , Dardani Ian , Jiang Connie , Goff Loyal A. , Dölen Gül
Published: March 2020

Parallel processing circuits are thought to dramatically expand the network capabilities of the nervous system. Magnocellular and parvocellular oxytocin neurons have been proposed to subserve two parallel streams of social information processing, which allow a single molecule to encode a diverse array of ethologically distinct behaviors, although to date direct evidence to support this hypothesis is lacking. Here we provide the first comprehensive characterization of magnocellular and parvocellular oxytocin neurons, validated across anatomical, projection target, electrophysiological, and transcriptional criteria. We next used novel multiple feature selection tools inFmr1KO mice to provide direct evidence that normal functioning of the parvocellular but not magnocellular oxytocin pathway is required for autism-relevant social reward behavior. Finally, we demonstrate that autism risk genes are uniquely enriched in parvocellular oxytocin neurons. Taken together these results provide the first evidence that oxytocin pathway specific pathogenic mechanisms account for social impairments across a broad range of autism etiologies.

One Sentence Summary

Pathoclisis of parvocellular oxytocin neurons plays an important role in the pathogenesis of social impairments in autism.
When Darkness Becomes a Ray of Light in the Dark Times: Understanding the COVID-19 via the Comparative Analysis of the Dark Proteomes of SARS-CoV-2, Human SARS and Bat SARS-Like Coronaviruses
Authors: Giri Rajanish , Bhardwaj Taniya , Shegane Meenakshi , Gehi Bhuvaneshwari R. , Kumar Prateek , Gadhave Kundlik , Oldfield Christopher J. , Uversky Vladimir N.
Published: March 2020

Recently emerged coronavirus designated as SARS-CoV-2 (also known as 2019 novel coronavirus (2019-nCoV) or Wuhan coronavirus) is a causative agent of coronavirus disease 2019 (COVID-19), which is rapidly spreading throughout the world now. More than 9,00,000 cases of SARS-CoV-2 infection and more than 47,000 COVID-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. World Health Organization (WHO) has declared the SARS-CoV-2 spread as a global public health emergency and admitted that the COVID-19 is a pandemic now. The multiple sequence alignment data correlated with the already published reports on the SARS-CoV-2 evolution and indicated that this virus is closely related to the bat Severe Acute Respiratory Syndrome-like coronavirus (bat SARS-like CoV) and the well-studied Human SARS coronavirus (SARS CoV). The disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. Therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of SARS-CoV-2, bat SARS-like, and human SARS CoVs by analysing the prevalence of intrinsic disorder in their proteins. According to our findings, SARS-CoV-2 proteome contains very significant levels of structural order. In fact, except for Nucleocapsid, Nsp8, and ORF6, the vast majority of SARS-CoV-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (IDPRs). However, IDPRs found in SARS-CoV-2 proteins are functionally important. For example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all SARS-CoV-2 proteins were shown to contain molecular recognition features (MoRFs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. The results of our extensive investigation of the dark side of the SARS-CoV-2 proteome will have important implications for the structural and non-structural biology of SARS or SARS-like coronaviruses.

Significance

The infection caused by a novel coronavirus (SARS-CoV-2) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current COVID-19 pandemic. No in-depth information on structures and functions of SARS-CoV-2 proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. Our study provides the first comparative analysis of the order- and disorder-based features of the SARS-CoV-2 proteome relative to human SARS and bat CoV that may be useful for structure-based drug discovery.
Arterial pulsations drive oscillatory flow of CSF but not directional pumping
Authors: Kedarasetti Ravi Teja , Drew Patrick J. , Costanzo Francesco
Published: March 2020

The brain lacks a traditional lymphatic system for metabolite clearance. The existence a “glymphatic system” where metabolites are removed from the brain’s extracellular space by convective exchange between interstitial fluid (ISF) and cerebrospinal fluid (CSF) along the paravascular spaces (PVS) around cerebral blood vessels has been controversial for nearly a decade. While recent work has shown clear evidence of directional flow of CSF in the PVS in anesthetized mice, the driving force for the observed fluid flow remains elusive. The heartbeat-driven peristaltic pulsation of arteries has been proposed as a probable driver of directed CSF flow. In this study, we use rigorous fluid dynamic simulations to provide a physical interpretation for peristaltic pumping of fluids. Our simulations match the experimental results and show that arterial pulsations only drive oscillatory motion of CSF in the PVS. The observed directional CSF flow can be explained by naturally occurring and/or experimenter-generated pressure differences.
Encoding Fear of Heights by Basolateral Amygdala Neurons
Authors: Liu Jun , Lin Longnian , Wang Dong V
Published: March 2020

Fear of heights is evolutionarily important for survival, yet it is unclear how and which brain regions encode such height threats. Given the importance of the basolateral amygdala (BLA) in processing both learned and innate fear, we investigated how BLA neurons may respond to high place exposure in freely behaving mice. We found that a discrete set of BLA neurons exhibited robust firing increases when the mouse was either exploring or placed on a high place, accompanied by increased heart rate and freezing. Importantly, these high-place fear neurons were only activated under height threats but not mild anxiogenic conditions. Furthermore, after a fear conditioning procedure, these high-place fear neurons developed conditioned responses to the context, but not the cue, indicating a convergence in encoding of dangerous/risky contextual information. Our results provide insights into the neural representation of the fear of heights and may have implications for treatment of excessive fear disorders.
Adaptive FDR thresholding of Fourier shell correlation for resolution estimation of cryo-EM maps
Authors: Beckers Maximilian , Sachse Carsten
Published: March 2020

Fourier shell correlation (FSC) has become a standard quantity for resolution estimation in electron cryo-microscopy. However, the resolution determination step is still subjective and not fully automated as it involves a series of map interventions before FSC computation and includes the selection of a common threshold. Here, we apply the statistical methods of permutation sampling and false discovery rate (FDR) control to the resolution-dependent correlation measure. The approach allows fully automated and mask-free resolution determination based on adaptive thresholding of FSC curves. We demonstrate the applicability for global, local and directional resolution estimation and show that the developed criterion termed FDR-FSC gives realistic resolution estimates based on a statistical significance criterion while eliminating the need of any map manipulations. The algorithms are implemented in a user-friendly GUI based software tool termed SPoC (https://github.com/MaximilianBeckers/SPOC).
Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells
Authors: Sui Lina , Xin Yurong , Georgieva Daniela , Diedenhofen Giacomo , Haataja Leena , Su Qi , Wang Yong , Zuccaro Michael , Kim Jinrang , Fu Jiayu , Xing Yuan , Baum Danielle , Goland Robin S. , Oberholzer Jose , Barbetti Fabrizio , Arvan Peter , Kleiner Sandra , Egli Dieter
Published: March 2020

Limitations in cell proliferation are a key barrier to reprogramming differentiated cells to pluripotent stem cells, and conversely, acquiring these limitations may be important to establish the differentiated state. The pancreas, and beta cells in particular have a low proliferative potential, which limits regeneration, but how these limitations are established is largely unknown. Understanding proliferation potential is important for the safty of cell replacement therapy with cell products made from pluripotent stem cell which have unlimited proliferative potential. Here we test a novel hypothesis, that these limitations are established through limitations in S-phase progression. We used a stem cell-based system to expose differentiating stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion. Upon release from these molecules, we determined growth potential, differentiation and function of insulin-producing endocrine cells bothin vitroand after graftingin vivo. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells, improved the stability of the differentiated state, and protected mice from diabetes without the formation of cystic growths. Therefore, a compromised ability to enter S-phase and replicate the genome is a functionally important property of pancreatic endocrine differentiation, and can be exploited to generate insulin-producing organoids with predictable growth potential after transplantation.
Notch effector Hes1 marks an early perichondrial population of skeletal progenitor cells at the onset of endochondral bone development
Authors: Matsushita Yuki , Nagata Mizuki , Welch Joshua D. , Wong Sunny Y. , Ono Wanida , Ono Noriaki
Published: March 2020

The perichondrium, a fibrous tissue surrounding the fetal cartilage, is an essential component of developing endochondral bones that provides a source of skeletal progenitor cells. However, perichondrial cells remain poorly characterized due to lack of knowledge on their cellular diversity and subset-specific mouse genetics tools. Single cell RNA-seq analyses reveal a contiguous nature of the fetal chondrocyte-perichondrial cell lineage that shares an overlapping set of marker genes. Subsequent cell-lineage analyses using multiplecreERlines active in fetal perichondrial cells –Hes1-creER,Dlx5-creER– and chondrocytes –Fgfr3-creER– illustrate their distinctive contribution to endochondral bone development; postnatally, these cells contribute to the functionally distinct bone marrow stromal compartments. Particularly, Notch effectorHes1-creERmarks an early skeletal progenitor cell population in the primordium that robustly populates multiple skeletal compartments. These findings support the concept that perichondrial cells participate in endochondral bone development through a distinct route, by providing a complementary source of skeletal progenitor cells.
Endothelial mTOR maintains hematopoiesis during aging
Authors: Ramalingam Pradeep , Poulos Michael G. , Gutkin Michael C. , Katsnelson Lizabeth , Freire Ana G. , Lazzari Elisa , Butler Jason M.
Published: March 2020

Aging leads to a decline in hematopoietic stem and progenitor cell (HSPC) function. We recently discovered that aging of bone marrow endothelial cells (BMECs) leads to an altered crosstalk between the BMEC niche and HSPCs, that instructs young HSPCs to behave as aged HSPCs. Here, we demonstrate aging leads to a decrease in mTOR signaling within BMECs that potentially underlies the age-related impairment of their niche activity. Our findings reveal that pharmacological inhibition of mTOR using Rapamycin has deleterious effects on hematopoiesis. To formally determine whether endothelial-specific inhibition of mTOR can influence hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that their HSPCs displayed attributes of an aged hematopoietic system. Transcriptional profiling of HSPCs from mTOR(ECKO)mice revealed that their transcriptome resembled aged HSPCs. Notably, during serial transplantations, exposure of wild type HSPCs to an mTOR(ECKO)microenvironment was sufficient to recapitulate aging-associated phenotypes, confirming the instructive role of EC-derived signals in governing HSPC aging.

Ramalingam et al. demonstrate that pharmacological inhibition of mTOR adversely impacts aging hematopoiesis. The authors demonstrate that aging results in decreased mTOR signaling within the bone marrow endothelium and endothelial-specific inhibition of mTOR causes hematopoietic defects observed during physiological aging.
ER-Mitochondria Contacts Promote Mitochondrial-Derived Compartment Biogenesis
Authors: English Alyssa M. , Kornmann Benoît , Shaw Janet M. , Hughes Adam L.
Published: March 2020

Mitochondria are dynamic organelles with essential roles in signaling and metabolism. We recently identified a new cellular structure called the mitochondrial-derived compartment (MDC) that is generated from mitochondria in response to amino acid elevation. MDCs protect cells from amino acid toxicity, but how cells form MDCs is unclear. Here, we show that MDCs are micron-sized, lumen-containing organelles that form at sites of contact between the ER and mitochondria. Upon formation, MDCs stably persist at ER-mitochondria contacts for extended periods of time. MDC formation requires the ER-mitochondria encounter structure (ERMES) and GTP hydrolysis by the conserved GTPase Gem1. Unexpectedly, MDC formation is not linked to the role of ERMES/Gem1 in the maintenance of mitochondrial phospholipid homeostasis. Our results identify an important role for ER-mitochondria contacts in the biogenesis of MDCs.

Abbreviations used in this paper: ERMES, ER-mitochondria encounter structure; IMM, inner mitochondrial membrane; MDC, mitochondrial-derived compartment; OMM, outer mitochondrial membrane.

English et al. use super-resolution imaging to show that mitochondrial-derived compartments are lumen-containing organelles that form at sites of contact between the ER and mitochondria. Mitochondrial-derived compartment biogenesis requires a noncanonical function of the ERMES complex and the conserved GTPase Gem1.
Automated curation of CNMF-E-extracted ROI spatial footprints and calcium traces using open-source AutoML tools
Authors: Tran LM , Mocle AJ , Ramsaran AI , Jacob AD , Frankland PW , Josselyn SA
Published: March 2020

In vivo 1-photon calcium imaging is an increasingly prevalent method in behavioural neuroscience. Numerous analysis pipelines have been developed to improve the reliability and scalability of pre-processing and ROI extraction for these large calcium imaging datasets. Despite these advancements in pre-processing methods, manual curation of the extracted spatial footprints and calcium traces of neurons remains important for quality control. Here, we propose an additional semi-automated curation step for sorting spatial footprints and calcium traces from putative neurons extracted using the popular CNMF-E algorithm. We used the automated machine learning tools TPOT and AutoSklearn to generate classifiers to curate the extracted ROIs trained on a subset of human-labeled data. AutoSklearn produced the best performing classifier, achieving an F1 score > 92% on the ground truth test dataset. This automated approach is a useful strategy for filtering ROIs with relatively few labeled data points, and can be easily added to pre-existing pipelines currently using CNMF-E for ROI extraction.
CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses
Authors: Sakers Kristina , Liu Yating , Llaci Lorida , Vasek Michael J. , Rieger Michael A. , Brophy Sean , Tycksen Eric , Lewis Renate , Maloney Susan E. , Dougherty Joseph D.
Published: March 2020

Quaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization by the cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3’ UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocyte’s territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-typesin vivo.