bluuebunny/crossref_metadata_2025_split huggingface dataset:
Double-zero-event studies matter: a re-evaluation of physical distancing, face masks, and eye protection for preventing person-to-person transmission of COVID-19 and its policy impact
Authors: Xiao Mengli , Lin Lifeng , Hodges James S. , Xu Chang , Chu Haitao 
Published: August 2020

AbstractObjectivesHigh-quality meta-analyses on COVID-19 are in urgent demand for evidence-based decision making. However, conventional approaches exclude double-zero-event studies (DZS) from meta-analyses. We assessed whether including such studies impacts the conclusions in a recent systematic urgent review on prevention measures for preventing person-to-person transmission of COVID-19.Study designs and settingsWe extracted data for meta-analyses containing DZS from a recent review that assessed the effects of physical distancing, face masks, and eye protection for preventing person-to-person transmission. A bivariate generalized linear mixed model was used to re-do the meta-analyses with DZS included. We compared the synthesized relative risks (RRs) of the three prevention measures, their 95% confidence intervals (CI), and significance tests (at the level of 0.05) including and excluding DZS.ResultsThe re-analyzed COVID-19 data containing DZS involved a total of 1,784 participants who were not considered in the original review. Including DZS noticeably changed the synthesized RRs and 95% CIs of several interventions. For the meta-analysis of the effect of physical distancing, the RR of COVID-19 decreased from 0.15 (95% CI, 0.03 to 0.73) to 0.07 (95% CI, 0.01 to 0.98). For several meta-analyses, the statistical significance of the synthesized RR was changed. The RR of eye protection with a physical distance of 2 m and the RR of physical distancing when using N95 respirators were no longer statistically significant after including DZS.ConclusionsDZS may contain useful information. Sensitivity analyses that include DZS in meta-analysis are recommended.
Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins
Authors: Gerson Julia E. , Linton Hunter , Xing Jiazheng , Sutter Alexandra B. , Kakos Fayth S. , Ryou Jaimie , Liggans Nyjerus , Sharkey Lisa M. , Safren Nathaniel , Paulson Henry L. , Ivanova Magdalena I. 
Published: August 2020

AbstractThe brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown.We report that the three ubiquilins differ in aggregation propensity, revealed byin-vitroexperiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrilsin vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by the brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements.We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and levels of aggregation. This variability among ubiquilins along the continuum from liquid-like to solid likely informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in various neurodegenerative diseases.
An easy and reliable whole blood freezing method for flow cytometry immunophenotyping and functional analyses
Authors: Braudeau Cecile , Guen Nina Salabert-Le , Justine Chevreuil , Marie Rimbert , Martin Jerome C. , Josien Regis 
Published: August 2020

ABSTRACTBackgroundImmune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods.MethodsIn this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor®CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.ResultsThough partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa).ConclusionsIn conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.
Functional Evidence for Biased Inhibition of G protein Signaling by YM-254890 in Human Coronary Artery Endothelial Cells
Authors: Peng Qianman , Alqahtani Saud , Nasrullah Mohammed Zahid A , Shen Jianzhong 
Published: August 2020

ABSTRACTSmall molecular chemicals targeting individual subtype of G proteins including Gs, Gi/o and Gq has been lacking, except forpertussis toxinbeing an established selective peptide inhibitor of the Gi/o protein. Recently, a cyclic depsipeptide compound YM-254890 isolated from culture broth ofChromobacterium sp. was reported as a selective inhibitor for the Gq protein by blocking GDP exchange of GTP on the α subunit of Gq complex. However, functional selectivity of YM-254890 towards various G proteins was not fully characterized, primarily due to its restricted availability before 2017. Here, using human coronary artery endothelial cells as a model, we performed a systemic pharmacological evaluation on the functional selectivity of YM-254890 on multiple G protein-mediated receptor signaling. First, we confirmed that YM-254890, at 30 nM, abolished UTP-activated P2Y2 receptor-mediated Ca2+signaling and ERK1/2 phosphorylation, indicating its potent inhibition on the Gq protein. However, we unexpectedly found that YM-254890 also significantly suppressed cAMP elevation and ERK1/2 phosphorylation induced by multiple Gs-coupled receptors including β2-adrenegic, adenosine A2and PGI2receptors. Surprisingly, although YM-254890 had no impact on CXCR4/Gi/o protein-mediated suppression of cAMP production, it abolished ERK1/2 activation. Further, no cellular toxicity was observed for YM-254890, and it neither affected A23187- or thapsigargin-induced Ca2+signaling, nor forskolin-induced cAMP elevation and growth factor-induced MAPK signaling. We conclude that YM-254890 is not a selective inhibitor for Gq protein; instead, it acts as a broad spectrum inhibitor for Gq and Gs proteins and exhibits a biased inhibition on Gi/o signaling, without affecting non-GPCR-mediated cellular signaling.
Efferent retargeting in above-knee amputees is positively related to phantom limb pain
Authors: Therrien Amanda S. , Howard Cortney , Buxbaum Laurel J. 
Published: August 2020

AbstractMany individuals who undergo limb amputation experience persistent phantom limb pain (PLP). The underlying mechanism of PLP is unknown, but the phenomenon has been associated with reorganization in sensorimotor cortex following amputation. The traditional view is that cortical reorganization degrades the missing limb’s representation. However, recent work suggests that an amputated limb’s cortical representation remains intact and that reorganization reflects a retargeting of efferent projections to residual muscles proximal to the amputation site. Evidence of retargeting has only been shown in individuals with upper limb amputations, and the relationship of retargeting to PLP is controversial. This study assessed retargeting and its relationship to PLP in 10 individuals with lower limb amputations. We recorded electromyographic (EMG) activity in a residual thigh muscle (vastus lateralis, VL) in patients with above-knee amputations during cyclical movements of the foot. VL activity on the amputated side was compared to that recorded on patients’ intact side while they moved their phantom and intact feet, respectively. VL activity in the patient group was also compared to VL activity from a sample of 9 control participants with no amputation. We show that phantom foot movement is associated with greater VL activity in the amputated leg than that seen in the intact leg as well as that exhibited by controls. The magnitude of residual VL activity was also positively related to ratings of PLP. These results provide the first support for retargeting in lower limb amputees and suggest that retargeting is related to the experience of phantom pain.New and NoteworthyPrevious work has only examined retargeting in upper limb amputees. This study provides evidence for retargeting in lower limb amputees and suggests that retargeting is related to phantom limb pain.
Matching a snail’s pace: Successful use of environmental DNA techniques to detect early stages of invasion by the destructive New Zealand mud snail
Authors: Woodell James D. , Neiman Maurine , Levri Edward P. 
Published: August 2020

ABSTRACTEarly detection of invasive species allows for a more rapid and effective response. Restoration of the native ecosystem after an invasive population has established is expensive and difficult but more likely to succeed when invasions are detected early in the invasion process. Containment efforts to prevent the spread of known invasions also benefit from earlier knowledge of invaded sites. Environmental DNA (eDNA) techniques have emerged as a tool that can identify invasive species at a distinctly earlier time point than traditional methods of detection. Due to expected range expansion in eastern North America, we focus on the destructive New Zealand Mud SnailPotamopyrgus antipodarum(NZMS) invasion. We collected water samples from eight sites that prior evidence indicated were not yet invaded by the NZMS. After filtering these samples to collect eDNA, we used a species-specific probe with qPCR to identify NZMS eDNA. We found evidence for NZMS invasion at five of the eight sites, with later physical confirmation of mud snails at one of these sites. This study is the first example of successful detection of a previously unidentified invasive population of NZMS, setting the stage for further monitoring of at-risk sites to detect and control new invasions of this destructive snail. This study also shows potential opportunities for invasion monitoring offered by using low-cost efforts and methods that are adaptable for citizen science.
Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate
Authors: Addetia Amin , Crawford Katharine HD , Dingens Adam , Zhu Haiying , Roychoudhury Pavitra , Huang Meei-Li , Jerome Keith R. , Bloom Jesse D. , Greninger Alexander L. 
Published: August 2020

AbstractThe development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have only been performed in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral RT-PCR testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR positive viral test with Ct <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested the outbreak originated largely from a single viral clade. Only three crewmembers tested seropositive prior to the boat’s departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of these crewmembers with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against re-infection (Fisher’s exact test, p=0.002).
LAMP-BEAC: Detection of SARS-CoV-2 RNA Using RT-LAMP and Molecular Beacons
Authors: Sherrill-Mix Scott , Hwang Young , Roche Aoife M. , Glascock Abigail , Weiss Susan R. , Li Yize , Haddad Leila , Deraska Peter , Monahan Caitlin , Kromer Andrew , Graham-Wooten Jevon , Taylor Louis J. , Abella Benjamin S. , Ganguly Arupa , Collman Ronald G. , Van Duyne Gregory D. , Bushman Frederic D. 
Published: August 2020

AbstractBackgroundRapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse Transcription and Loop-Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.ResultsHere we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.ConclusionsLAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.
Broiler Chickens and Early Life Programming: Microbiome transplant-induced cecal bacteriome dynamics and phenotypic effects
Authors: Ramírez Gustavo A. , Richardson Ella , Clark Jory , Keshri Jitendra , Drechsler Yvonne , Berrang Mark E. , Meinersmann Richard J. , Cox Nelson A. , Oakley Brian B. 
Published: August 2020

AbstractThe concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first derive a stable source of inoculum. Subsequently, we compared microbiome development between chicks receiving this passaged cecal material, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa were identified across treatment types that may inform future studies aimed at identifying strains associated with beneficial phenotypes.
Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion
Authors: Papa Guido , Mallery Donna L. , Albecka Anna , Welch Lawrence , Cattin-Ortolá Jérôme , Luptak Jakub , Paul David , McMahon Harvey T. , Goodfellow Ian G. , Carter Andrew , Munro Sean , James Leo C. 
Published: August 2020

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.
CRISPR-Decryptr reveals cis-regulatory elements from noncoding perturbation screens
Authors: Rasmussen Anders , Äijö Tarmo , Gabitto Mariano Ignacio , Carriero Nicholas , Sanjana Neville , Skok Jane , Bonneau Richard 
Published: August 2020

AbstractClustered Regularly Interspace Short Palindromic Repeats (CRISPR)-Cas9 genome editing methods provide the tools necessary to examine phenotypic impacts of targeted perturbations in high-throughput screens. While these technologies have the potential to reveal functional elements with direct therapeutic applications, statistical techniques to analyze noncoding screen data remain limited. We present CRISPR-Decryptr, a computational tool for the analysis of CRISPR noncoding screens. Our method leverages experimental design: accounting for multiple conditions, controls, and replicates to infer the regulatory landscape of noncoding genomic regions. We validate our method on a variety of mutagenesis, CRISPR activation, and CRISPR interference screens, extracting new insights from previously published data.
Characterisation of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification
Authors: Baddock Hannah T. , Brolih Sanja , Yosaatmadja Yuliana , Ratnaweera Malitha , Bielinski Marcin , Swift Lonnie P. , Cruz-Migoni Abimael , Morris Garrett M. , Schofield Christopher J. , Gileadi Opher , McHugh Peter J. 
Published: August 2020

AbstractThe SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3’-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.
The SARS-CoV-2 Spike harbours a lipid binding pocket which modulates stability of the prefusion trimer
Authors: Carrique Loic , Duyvesteyn Helen ME , Malinauskas Tomas , Zhao Yuguang , Ren Jingshan , Zhou Daming , Walter Thomas S , Radecke Julika , Huo Jiandong , Ruza Reinis R , Shah Pranav NM , Fry Elizabeth E , Stuart David I 
Published: August 2020

SummaryLarge trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is ‘locked’ into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the ‘locked’ form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the ‘locked’ form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies.HighlightsSARS-CoV-2 Spike can adopt a ‘locked’ conformation with all receptor binding domains (RBDs) down, likely to represent the prefusion resting stateThis ‘locked’ conformation is compact and stable, braced by lipid bound within a potentially druggable pocketKey neutralization epitopes are shielded in the ‘locked’ formLoss of lipid may trigger a cascade of events that lead to cell entry analogous to the role of lipids in enterovirus cell entry