bluuebunny/crossref_metadata_2025_split huggingface dataset:
Compounds enhancing human sperm motility identified using a high-throughput phenotypic screening platform
Authors: Gruber Franz S. , Johnston Zoe C. , Norcross Neil R. , Georgiou Irene , Wilson Caroline , Read Kevin D. , Gilbert Ian H. , Swedlow Jason R. , de Silva Sarah Martins , Barratt Christopher LR 
Published: September 2021

AbstractStudy questionCan a high-throughput screening platform facilitate male fertility drug discovery?Summary answerA high-throughput screening platform identified a large number of compounds that enhanced sperm motility.What is known alreadySeveral efforts to find small molecules modulating sperm function have been performed but not using high-throughput technology.Study design, size, durationHealthy donor semen samples were used and samples were pooled (3-5 donors per pool). Primary screening was performed in singlicate; dose-response screening was performed in duplicate (independent donor pools).Participants/materials, setting, methodsSpermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 uM) to identify those compounds with enhancing effects on motility. A total of ∼17,000 compounds from the following libraries: ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box were screened. Dose response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a University setting.Main results and the role of chanceFrom our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to DMSO treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified in particular PDE10A inhibitors as well as number of compounds not previously identified/known to enhance human sperm motility such as those related to GABA signaling.Limitations, reasons for cautionCompounds have been tested with prepared donor spermatozoa and only incubated for a short period of time. Therefore, the effect of compounds on whole semen or with longer incubation time may be different. All experiments were performed in vitro.Wider implications of the findingsThis phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential inhibitors as promising start-point for a medicinal chemistry programme for potential enhancement of male infertility. Moreover, with disclosure of the results of screening we present a substantial resource to inform further work in the fieldStudy funding/competing interest(s)This study was supported by the Bill and Melinda Gates Foundation and Scottish Funding Council and Scottish Universities Life Science Alliance.
MAGI-MS: Multiple seed-centric module discovery
Authors: Chow Julie C. , Zhou Ryan , Hormozdiari Fereydoun 
Published: September 2021

AbstractComplex disorders manifest by the interaction of multiple genetic and environmental factors. Through the construction of genetic modules that consist of highly co-expressed genes, it is possible to identify genes that participate in common biological pathways relevant to specific phenotypes. We have previously developed tools MAGI and MAGI-S for genetic module discovery by incorporating co-expression and protein-interaction networks. Here we introduce an extension to MAGI-S, denoted as Merging Affected Genes into Integrated Networks - Multiple Seeds (MAGI-MS), that permits the user to further specify a disease pathway of interest by selecting multiple seed genes likely to function in the same molecular mechanism. By providing MAGI-MS with pairs of seed genes involved in processes underlying certain classes of neurodevelopmental disorders, such as epilepsy, we demonstrate that MAGI-MS can reveal modules enriched in genes relevant to chemical synaptic transmission, glutamatergic synapse, and other functions associated with the provided seed genes.Availability and implementationMAGI-MS is free and is available at: https://github.com/jchow32/MAGI-MS
The role of antigen recognition in the γδ T cell response at the controlled stage ofM. tuberculosisinfection
Authors: Chowdhury Roshni Roy , Valainis John R. , Kask Oliver , Ohanyan Mane , Sun Meng , Huang Huang , Dubey Megha , von Boehmer Lotta , Sola Elsa , Huang Xianxi , Nguyen Patricia K. , Scriba Thomas J. , Davis Mark M. , Bendall Sean C. , Chien Yueh-hsiu 
Published: September 2021

SummaryγδT cells contribute to host immune defense uniquely; but how they function in different stages (e.g., acute versus chronic) of a specific infection remains unclear. As the role ofγδT cells in early, activeMycobacterium tuberculosis(Mtb) infection is well documented, we focused on elucidating theγδT cell response in persistent or controlled Mtb infection. Systems analysis of circulatingγδT cells from a South African adolescent cohort identified a distinct population of CD8+γδT cells that expanded in this state. These cells had features indicative of persistent antigenic exposure but were robust cytolytic effectors and cytokine/chemokine producers. While theseγδT cells displayed an attenuated response to TCR-mediated stimulation, they expressed Natural Killer (NK) cell receptors and had robust CD16 (FcγRIIIA)-mediated cytotoxic response, suggesting alternative ways forγδT cells to control this stage of the infection. Despite this NK- like functionality, the CD8+γδT cells consisted of highly expanded clones, which utilized TCRs with different Vγ/δpairs. Theses TCRs could respond to an Mtb-lysate, but not to phosphoantigens, which are components of Mtb-lysate that activateγδT cells in acute Mtb infection, indicating that the CD8+γδT cells were induced in a stage-specific, antigen-driven manner. Indeed, trajectory analysis showed that theseγδT cells arose from naive cells that had traversed distinct differentiation paths in this infection stage. Importantly, increased levels of CD8+γδT cells were also found in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may lead to similarγδT cell responses.
Humoral immune responses against seasonal coronaviruses predict efficiency of SARS-CoV-2 spike targeting, FcγR activation, and corresponding COVID-19 disease severity
Authors: Garrido Jose L. , Medina Matias , Bravo Felipe , McGee Sarah , Fuentes Francisco , Calvo Mario , Bowman James W. , Bahl Christopher D. , Barría Maria Inés , Brachman Rebecca A. , Alvarez Raymond A. 
Published: September 2021

AbstractDespite SARS-CoV-2 being a “novel” coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (FcγR2a & FcγR3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2’FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2’FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.
The 3D spatial constraint on 6.1 million amino acid sites in the human proteome
Authors: Li Bian , Roden Dan M. , Capra John A. 
Published: September 2021

AbstractQuantification of the tolerance of protein-coding sites to genetic variation within human populations has become a cornerstone of the prediction of the function of genomic variants. We hypothesize that the constraint on missense variation at individual amino acid sites is largely shaped by direct 3D interactions with neighboring sites. To quantify the constraint on protein-coding genetic variation in 3D spatial neighborhoods, we introduce a new framework called COntact Set MISsense tolerance (or COSMIS) for estimating constraint. Leveraging recent advances in computational structure prediction, large-scale sequencing data from gnomAD, and a mutation-spectrum-aware statistical model, we comprehensively map the landscape of 3D spatial constraint on 6.1 amino acid sites covering >80% (16,533) of human proteins. We show that the human proteome is broadly under 3D spatial constraint and that the level of spatial constraint is strongly associated with disease relevance both at the individual site level and the protein level. We demonstrate that COSMIS performs significantly better at a range of variant interpretation tasks than other population-based constraint metrics while also providing biophysical insight into the potential functional roles of constrained sites. We make our constraint maps freely available and anticipate that the structural landscape of constrained sites identified by COSMIS will facilitate interpretation of protein-coding variation in human evolution and prioritization of sites for mechanistic or functional investigation.
Mechanistic insights into dynamic mutual regulation of USP14 and proteasome
Authors: Zhang Shuwen , Zou Shitao , Yin Deyao , Finley Daniel , Wu Zhaolong , Mao Youdong 
Published: September 2021

AbstractProteasomal degradation of ubiquitylated proteins is sophisticatedly regulated at multiple levels1–3. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme USP14 that associates reversibly with the proteasome. How USP14 is activated and regulates the proteasome function remains unknown4–7. Here we report cryo-electron microscopy (cryo-EM) structures of human USP14 in complex with the 26S proteasome in nine conformational states at 3.0-3.6 Å resolution, captured during polyubiquitylated protein degradation. Time-resolved cryo-EM analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14 and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, USP14 activation allosterically reprograms conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate8–10, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation11–13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide unprecedented insights into the complete functional cycles of USP14-regulated proteasome and of USP14 activation-deubiquitylation-disassembly and establish mechanistic foundations for USP14-targeted therapeutic discovery.
Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing
Authors: Marquet Mike , Zöllkau Janine , Pastuschek Jana , Viehweger Adrian , Schleußner Ekkehard , Makarewicz Oliwia , Pletz Mathias W. , Ehricht Ralf , Brandt Christian 
Published: September 2021

AbstractMetagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore’s adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective ‘human host depletion’ resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.
Transitioning to confined spaces impacts bacterial swimming and escape response
Authors: Lynch Jonathan B. , James Nicholas , McFall-Ngai Margaret , Ruby Edward G. , Shin Sangwoo , Takagi Daisuke 
Published: September 2021

AbstractSymbiotic bacteria often navigate complex environments before colonizing privileged sites in their host organism. Chemical gradients are known to facilitate directional taxis of these bacteria, guiding them towards their eventual destination. However, less is known about the role of physical features in shaping the path the bacteria take and defining how they traverse a given space. The flagellated marine bacteriumVibrio fischeri,which forms a binary symbiosis with the Hawaiian bobtail squid,Euprymna scolopes, must navigate tight physical confinement, squeezing through a bottleneck constricting to ~2 μm in width on the way to its eventual home. Using microfluidicin vitroexperiments, we discovered thatV. fischericells alter their behavior upon entry into confined space, straightening their swimming paths and promoting escape from confinement. Using a computational model, we attributed this escape response to two factors: reduced directional fluctuation and a refractory period between reversals. Additional experiments in asymmetric capillary tubes confirmed thatV. fischeriquickly escape from tapered ends, even when drawn into the ends by chemoattraction. This avoidance was apparent down to a limit of confinement approaching the diameter of the cell itself, resulting in a balance between chemoattraction and evasion of physical confinement. Our findings demonstrate that non-trivial distributions of swimming bacteria can emerge from simple physical gradients in the level of confinement. Tight spaces may serve as an additional, crucial cue for bacteria while they navigate complex environments to enter specific habitats.Significance StatementSymbiotic bacteria that navigate to and through specific host tissues often face tight physical confinement. This work reveals that confinement-associated changes in swimming can dramatically alter taxis, shaping bacterial localization in conjuncture with other motility-directing cues. This work helps explain how bacteria can avoid getting stuck in confined areas while transiting to privileged spaces, adding confinement as an environmental cue that symbiotic bacteria use to shape their motility behavior.
Muscle weakness precedes atrophy during cancer cachexia and is associated with muscle-specific mitochondrial stress
Authors: Delfinis Luca J. , Bellissimo Catherine A. , Gandhi Shivam , DiBenedetto Sara N. , Rosa-Caldwell Megan E. , Rahman Fasih A. , Wiggs Michael P. , Schlattner Uwe , Quadrilatero Joe , Greene Nicholas P. , Perry Christopher G.R. 
Published: September 2021

AbstractMuscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but distinct responses between muscles and across time remains unclear. We aimed to determine the time-dependent and muscle-specific responses to Colon-26 (C26) cancer-induced cachexia in mice. At 2 weeks post-inoculation, the presence of small tumours did not alter body or muscle mass but decreased force production in the quadriceps and diaphragm. Pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H2O2 emission was elevated in diaphragm. At 4 weeks, large tumours corresponded to lower body mass, muscle mass, and cross-sectional area of fibers in quadriceps and diaphragm. Force production in quadriceps was unchanged but remained lower in diaphragm vs control. Mitochondrial respiration was increased while H2O2 emission was unchanged in both muscles vs control. Mitochondrial creatine sensitivity was compromised in quadriceps. These findings indicate muscle weakness precedes atrophy in quadriceps and diaphragm but is linked to heterogeneous mitochondrial alterations. Eventual muscle-specific restorations in force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscles to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.
Identification of the safe harbor locus, AAVS1, from porcine genome and site-specific integration of recombinase-mediated cassette exchange system in porcine fibroblasts using CRISPR/Cas9
Authors: Lee Choongil , Yum Soo-Young , Choi Woojae , Kim Seokjoong , Jang Goo , Koo Okjae 
Published: September 2021

AbstractGene integration at site-specific loci, such as safe harbor regions for stable expression via transgenesis, is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor site for human and mouse studies. In the present study, we found an AAVS1-like sequence in the porcine genome using the UCSC Genome Browser and designed TALEN and CRISPR/Cas9 to target AAVS1. The efficiency of CRISPR/Cas9 for targeting the AAVS1 locus in porcine cells was superior to that of TALEN. An AAVS1-targeting donor vector containing GFP was designed and cloned. We added a loxP-lox2272 cassette sequence to the donor vector for further exchange of various transgenes in the AAVS1-targeted cell line. The donor vector and CRISPR/Cas9 components targeting AAVS1 were transfected into a porcine fibroblast cell line. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in at the AAVS1 locus was confirmed by PCR analysis. To induce recombinase-mediated cassette exchange (RMCE), another donor vector containing the loxP-lox2272 cassette and inducible (Tet-on) Cre recombinase was cloned. The Cre-donor vector was transfected into the AAVS1-targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR analysis. In conclusion, gene targeting at the AAVS1 locus and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.
Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11
Authors: Dillard Kaylee E. , Terrace Cynthia , Javanmardi Kamyab , Kim Wantae , Forsberg Kevin J. , Finkelstein Ilya J. 
Published: September 2021

AbstractMobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but are generally specific for a narrow subset of Cas nucleases that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits diverse Cas9 sub-typesin vitroand human cells. Single-molecule fluorescence imaging reveals that AcrIIA11 interferes with the first steps of target search by reducingS. aureusCas9’s diffusion on non-specific DNA. DNA cleavage is inhibited because the AcrIIA11:Cas9 complex is kinetically trapped at PAM-rich decoy sites, preventing Cas9 from reaching its target. This work establishes that DNA trapping can be used to inhibit a broad spectrum of Cas9 orthologsin vitroand during mammalian genome editing.
The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and Cilia to Regulate Ciliogenesis and Ciliary Protein Traffic
Authors: Turn Rachel E. , Hu Yihan , Dewees Skylar I. , Devi Narra , East Michael P. , Hardin Katherine R. , Khatib Tala , Linnert Joshua , Wolfrum Uwe , Lim Michael J. , Casanova James E. , Caspary Tamara , Kahn Richard A. 
Published: September 2021

ABSTRACTELMODs are a family of three mammalian paralogs that display GTPase activating protein (GAP) activity towards a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogs ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon expression of activating mutants of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions. Thus, we believe that ELMOD1 and ELMOD3 perform multiple functions in cells, most prominently linked to ciliary biology and Golgi-ciliary traffic, and likely acting from more than one cellular location.