bluuebunny/crossref_metadata_2025 huggingface dataset:
Polycystin-2 ciliary extracellular vesicle release and targeting
Authors: Wang Juan , Nikonorova Inna A. , Gu Amanda , Sternberg Paul W. , Barr Maureen M.
Published: April 2020

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1]. An urgent need in the field is a fundamental understanding of physiological mechanisms driving EV generation and function. Ciliary EVs act as signaling devices inChlamydomonasandC. elegans[2–4]. Mammalian cilia shed EVs to eliminate unwanted receptors [5] or to retract cilia before entering the cell cycle [6]. Here we used our establishedC. elegansmodel to study sensory-evoked ciliary EV release and targeting using a fluorescently labeled EV cargo polycystin-2 (PKD-2). InC. elegansand mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation [7]. We find that males deposit PKD-2-carrying EVs onto the vulva of the hermaphrodite during mating. We also show that mechanical stimulation triggers release of PKD-2-carrying EVs from cilia. To our knowledge this is the first report of mechanoresponsive nature of the ciliary EV release and of ciliary EV directional transfer from one animal to another animal. Since the polycystins are evolutionarily conserved ciliary EV cargoes, our findings suggest that similar mechanisms for EV release and targeting may occur in other systems and biological contexts.
Sex, androgens and regulation of pulmonary AR, TMPRSS2 and ACE2
Authors: Baratchian Mehdi , McManus Jeffrey M. , Berk Mike , Nakamura Fumihiko , Mukhopadhyay Sanjay , Xu Weiling , Erzurum Serpil , Drazba Judy , Peterson John , Klein Eric A. , Gaston Ben , Sharifi Nima
Published: April 2020

The sex discordance in COVID-19 outcomes has been widely recognized, with males generally faring worse than females and a potential link to sex steroids. A plausible mechanism is androgen-induced expression of TMPRSS2 and/or ACE2 in pulmonary tissues that may increase susceptibility or severity in males. This hypothesis is the subject of several clinical trials of anti-androgen therapies around the world. Here, we investigated the sex-associated TMPRSS2 and ACE2 expression in human and mouse lungs and interrogated the possibility of pharmacologic modification of their expression with anti-androgens. We found no evidence for increasedTMPRSS2expression in the lungs of males compared to females in humans or mice. Furthermore, in male mice, treatment with the androgen receptor antagonist enzalutamide did not decrease pulmonary TMPRSS2. On the other hand, ACE2 and AR expression was sexually dimorphic and higher in males than females. ACE2 was moderately suppressible with enzalutamide therapy. Our work suggests that sex differences in COVID-19 outcomes attributable to viral entry are independent of TMPRSS2. Modest changes in ACE2 could account for some of the sex discordance.
Human iPSC-Derived Cardiomyocytes are Susceptible to SARS-CoV-2 Infection
Authors: Sharma Arun , Garcia Gustavo , Arumugaswami Vaithilingaraja , Svendsen Clive N.
Published: April 2020

Coronavirus disease 2019 (COVID-19) is a viral pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is predominantly defined by respiratory symptoms, but cardiac complications including arrhythmias, heart failure, and viral myocarditis are also prevalent. Although the systemic ischemic and inflammatory responses caused by COVID-19 can detrimentally affect cardiac function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is not well-understood. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to examine the mechanisms of cardiomyocyte-specific infection by SARS-CoV-2. Microscopy and immunofluorescence demonstrated that SARS-CoV-2 can enter and replicate within hiPSC-CMs, localizing at perinuclear locations within the cytoplasm. Viral cytopathic effect induced hiPSC-CM apoptosis and cessation of beating after 72 hours of infection. These studies show that SARS-CoV-2 can infect hiPSC-CMsin vitro, establishing a model for elucidating the mechanisms of infection and potentially a cardiac-specific antiviral drug screening platform.
Sequential CRISPR gene editing in human iPSCs charts the clonal evolution of leukemia
Authors: Wang Tiansu , Pine Allison R , Wesely Josephine , Yuan Han , Zamparo Lee , Kotini Andriana G , Leslie Christina , Papapetrou Eirini P
Published: April 2020

Human cancers arise through an evolutionary process whereby cells acquire somatic mutations that drive them to outgrow normal cells and create successive clonal populations. “Bottom-up” human cancer evolution models could help illuminate this process, but their creation has faced significant challenges. Here we combined human induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies to develop a model of the clonal evolution of acute myeloid leukemia (AML). Through the sequential introduction of 3 disease-causing mutations (ASXL1 C-terminus truncation, SRSF2P95L and NRASG12D), we obtained single, double and triple mutant iPSC lines that, upon hematopoietic differentiation, exhibit progressive dysplasia with increasing number of mutations, capturing distinct premalignant stages, including clonal hematopoiesis, myelodysplastic syndrome, and culminating in a transplantable leukemia. iPSC-derived clonal hematopoietic stem/progenitor cells recapitulate transcriptional and chromatin accessibility signatures of normal and malignant hematopoiesis found in primary human cells. By mapping dynamic changes in transcriptomes and chromatin landscapes, we characterize transcriptional programs driving specific stage transitions and identify vulnerabilities for early therapeutic targeting. Such synthetic “de novo oncogenesis” models can empower the investigation of multiple facets of the malignant transformation of human cells.
Artificial intelligence predicts the immunogenic landscape of SARS-CoV-2: toward universal blueprints for vaccine designs
Authors: Malone Brandon , Simovski Boris , Moliné Clément , Cheng Jun , Gheorghe Marius , Fontenelle Hugues , Vardaxis Ioannis , Tennøe Simen , Malmberg Jenny-Ann , Stratford Richard , Clancy Trevor
Published: April 2020

The global population is at present suffering from a pandemic of Coronavirus disease 2019 (COVID-19), caused by the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The goals of this study were to use artificial intelligence (AI) to predict blueprints for designing universal vaccines against SARS-CoV-2, that contain a sufficiently broad repertoire of T-cell epitopes capable of providing coverage and protection across the global population. To help achieve these aims, we profiled the entire SARS-CoV-2 proteome across the most frequent 100 HLA-A, HLA-B and HLA-DR alleles in the human population, using host-infected cell surface antigen presentation and immunogenicity predictors from theNEC Immune Profilersuite of tools, and generated comprehensive epitope maps. We then used these epitope maps as input for a Monte Carlo simulation designed to identify statistically significant “epitope hotspot” regions in the virus that are most likely to be immunogenic across a broad spectrum of HLA types. We then removed epitope hotspots that shared significant homology with proteins in the human proteome to reduce the chance of inducing off-target autoimmune responses. We also analyzed the antigen presentation and immunogenic landscape of all the nonsynonymous mutations across 3400 different sequences of the virus, to identify a trend whereby SARS-COV-2 mutations are predicted to have reduced potential to be presented by host-infected cells, and consequently detected by the host immune system. A sequence conservation analysis then removed epitope hotspots that occurred in less-conserved regions of the viral proteome. Finally, we used a database of the HLA genotypes of approximately 22 000 individuals to develop a “digital twin” type simulation to model how effective different combinations of hotspots would work in a diverse human population, and used the approach to identify an optimal constellation of epitopes hotspots that could provide maximum coverage in the global population. By combining the antigen presentation to the infected-host cell surface and immunogenicity predictions of theNEC Immune Profilerwith a robust Monte Carlo and digital twin simulation, we have managed to profile the entire SARS-CoV-2 proteome and identify a subset of epitope hotspots that could be harnessed in a vaccine formulation to provide a broad coverage across the global population.
Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons
Authors: Lindhout Feline W. , Kooistra Robbelien , Portegies Sybren , Herstel Lotte J. , Stucchi Riccardo , Snoek Basten L. , Altelaar Maarten , MacGillavry Harold D. , Wierenga Corette J. , Hoogenraad Casper C.
Published: April 2020

Early neuronal development is a well-coordinated process in which neuronal stem cells differentiate into polarized neurons. This process has been well studied in classical non-human model systems, but to what extent this is recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured human iPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. We found extensive remodeling of the neuron transcriptome and proteome, with altered mRNA expression of ~1,100 genes and different expression profiles of ~1,500 proteins during neuronal differentiation and polarization. We also identified a distinct stage in axon development marked by an increase in microtubule remodeling and apparent relocation of the axon initial segment from the distal to proximal axon. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.
One-pot Detection of COVID-19 with Real-time Reverse-transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay
Authors: Wang Deguo
Published: April 2020

Background

Rapid and reliable diagnostic assays were critical for prevention and control of the coronavirus pneumonia caused by COVID-19.

Objective

This study was to establish one-pot real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay and one-pot visual RT-LAMP assay for the detection of COVID-19.

Methods

Six specific LAMP primers targeting the N gene of COVID-19 were designed, the RT-LAMP reaction system was optimized with plasmid pUC57 containing N gene sequence, the detection limit was determined with a serial dilution of the plasmid pUC57 containing N gene sequence, and the one-pot real-time RT-LAMP assay and one-pot visual RT-LAMP assay for the detection of COVID-19 were established.

Results

Our results showed that the one-pot RT-LAMP assays can detect COVID-19 with a limit of ≥ 6 copies perμl−1of pUC57 containing N gene sequence.

Conclusion

This study provides rapid, reliable and sensitive tools for facilitating preliminary and cost-effective prevention and control of COVID-19.
Cellbow: a robust customizable cell segmentation program
Authors: Ren Huixia , Zhao Mengdi , Liu Bo , Yao Ruixiao , liu Qi , Ren Zhipeng , Wu Zirui , Gao Zongmao , Yang Xiaojing , Tang Chao
Published: April 2020

Time-lapse live cell imaging of a growing cell population is routine in many biological investigations. A major challenge in imaging analysis is accurate segmentation, a process to define the boundaries of cells based on raw image data. Current segmentation methods relying on single boundary features have problems in robustness when dealing with inhomogeneous foci which invariably happens in cell population imaging. Here, we demonstrated that combined with multi-layer training set strategy, a neural-network-based algorithm Cellbow can achieve accurate and robust segmentation of cells in broad and general settings. It can also facilitate long-term tracking of cell growth and division. Furthermore, Cellbow is customizable and generalizable. It is broadly applicable to segmenting fluorescent images of diverse cell types with no further training needed. For bright-field images, only a small set of sample images of the specific cell type from the user may be needed for training. To facilitate the application of Cellbow, we provide a website on which one can online test the software, as well as an ImageJ plugin for the user to visualize the performance before software installation.
Structural Basis of SARS-CoV-2 Spike Protein Priming by TMPRSS2
Authors: Hussain Mushtaq , Jabeen Nusrat , Amanullah Anusha , Baig Ayesha Ashraf , Aziz Basma , Shabbir Sanya , Raza Fozia
Published: April 2020

Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational details of complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike and to point candidate drug targets. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provide structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.
Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins
Authors: Lee Chanjae , Cox Rachael M. , Papoulas Ophelia , Horani Amjad , Drew Kevin , Devitt Caitlin C. , Brody Steven L. , Marcotte Edward M. , Wallingford John B.
Published: April 2020

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we usein vivoimaging to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity purification mass-spectrometry ofin vivointeraction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.
Investigating the effects of incremental conditioning and supplemental dietary tryptophan on the voluntary activity and behaviour of mid-distance training sled dogs
Authors: Robinson Eve , Templeman James R. , Thornton Emma , Croney Candace C. , Niel Lee , Shoveller Anna K.
Published: April 2020

Serotonin is a neurotransmitter synthesized by the amino acid tryptophan (Trp) that has the potential to impact the behaviour and activity of dogs. The objective of this study was to assess the effects of supplemental Trp and a 12-week incremental training regimen on the voluntary activity and behaviour of client-owned Siberian Huskies. Sixteen dogs were blocked for age, BW and sex and then randomly allocated to either the control or treatment group. Both groups were fed the same dry extruded diet; however, the treatment group were supplemented with Trp to achieve a Trp: large neutral amino acid ratio of 0.075:1. Once a week, a 5-minute video recording was taken immediately pre- and post- exercise to evaluate dogs’ behaviours. Activity monitors were used to record voluntary activity on both training and rest days. Linear regression analysis was used to assess the relationship between training week and time spent performing each behaviour. Additionally, a repeated measure mixed model was used to test differences between diet groups and training week for both behavioural and activity count data. The time spent performing agonistic behaviours prior to exercise was negatively associated with week for treatment dogs (β = −0.32, 95% CI [−0.55, −0.10], P < 0.05) and no change was observed for control dogs (β = −0.13, 95% CI [−0.41, 0.15], P > 0.10). Treatment did not have any effect on activity levels (P > 0.10). For all dogs, locomotive behaviours decreased prior to exercise as weeks progressed (P < 0.05), while run day voluntary activity depended on the distance run that day (P < 0.05). These data suggest that sled dogs experience an exercise-induced reduction in voluntary locomotion in response to both single bouts and repetitive bouts of exercise. Additionally, tryptophan supplementation may decrease agonistic behaviours, without having any effect on voluntary activity.
Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2
Authors: Kang Yuan-Lin , Chou Yi-Ying , Rothlauf Paul W. , Liu Zhuoming , Soh Timothy K. , Cureton David , Case James Brett , Chen Rita E. , Diamond Michael S. , Whelan Sean P. J. , Kirchhausen Tom
Published: April 2020

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric VSV containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or SARS-CoV-2 (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small molecule inhibitors of the main endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define new tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.
Helicobacter hepaticusas disease driver in a novel CD40-mediated model of colitis
Authors: Friedrich Verena , Forne Ignasi , Matzek Dana , Ring Diana , Popper Bastian , Jochum Lara , Spriewald Stefanie , Straub Tobias , Imhof Axel , Krug Anne , Stecher Bärbel , Brocker Thomas
Published: April 2020

Gut microbiota and the immune system are in constant exchange, which shapes both, host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40 – CD40L axis showed promising results as these molecules have been described to be deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals, which lack intestinal CD103+dendritic cells (DCs) and therefore cannot induce regulatory T (iTreg) cells due to a constitutive CD40-signal in CD11c+cells. These mice rapidly develop spontaneous fatal colitis with an increase of inflammatory IL-17+IFN-γ+Th17/Th1 and IFN-γ+Th1 cells. In the present study we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria and we identified a 60 kDa chaperonin GroEL (Hsp60) fromHelicobacter hepaticus(Hh) as the main specific antigen targeted in absence of iTregs. When rederived to a differentHh-free SPF-microbiota, mice showed few signs of disease without fatalities, but upon recolonization of mice withHhwe found rapid disease onset and the generation of inflammatory Th17/Th1 and Th1 cells in the colon. Thus, the present work identifies a major bacterial antigen and highlights the impact of specific microorganisms on modulating the host immune response and its role on disease onset, progression and outcome in this colitis model.
Bioinformatics Study on Structural Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) For Better Understanding the Vaccine Development
Authors: Gulzar Sumaira , Husssain Saqib
Published: April 2020

Novel coronavirus 2019 (2019-nCoV), also known as SARS-CoV-2), leads high morbidity and mortality in global epidemics. Four structural proteins (surface glycoprotein (QIQ22760.1), envelop glycoprotein (QIQ22762.1), nucleocapsid phosphoprotein (QIQ22768.1) and membrane glycoprotein (QIQ22763.1)) of SARS-CoV-2 are extracted from the NCBI database and further analyzed with ExPASy ProtParam tool. Lucien is the highest in envelope, surface and membrane glycoprotein that is an optimal environment for rapid virus fixation on host cell's surface to the receptor molecule. Transmembrane region prediction was performed by SOSUI server. For all structural proteins, except nucleocapsid Phosphoprotein, the trans-membrane prediction indicates that the virus can enter the host easily. Domain analysis was done by SMART tool. Domain information helps in the function of the viral protein. Lastly, the 3D structure prediction was carried out by Swiss Model and the result validation was achieved by PROCHECK. Such models are the starting point of the community for structural drug and vaccine designs as well as virtual computational screening.
Cryo-EM structures of inactive and Gi-coupled GABAB heterodimer
Authors: Mao Chunyou , Shen Cangsong , Li Chuntao , Shen Dan-Dan , Xu Chanjuan , Zhang Shenglan , Zhou Rui , Shen Qingya , Chen Li-Nan , Jiang Zhinong , Liu Jianfeng , Zhang Yan
Published: April 2020

Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of human full-length heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with agonist and positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8-3.0 Å. Cryo-EM analysis of the activated-GABAB–Gi1 complex revealed that Gi1 couples to the activated receptor primarily in three major conformations, one via GB1 TM and two via GB2 TM, respectively. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistically shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of Gi1. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting dimeric interface of GABAB receptor.
Long-run bacteria-phage coexistence dynamics under natural habitat conditions in an environmental biotechnology system
Authors: Guerrero Leandro D. , Pérez María V. , Orellana Esteban , Piuri Mariana , Quiroga Cecilia , Erijman Leonardo
Published: April 2020

Bacterial viruses are widespread and abundant across natural and engineered habitats. They influence ecosystem functioning through interactions with their hosts. Laboratory studies of phage-host pairs have advanced our understanding of phenotypic and genetic diversification in bacteria and phages. However, the dynamics of phage-host interactions has been seldom recorded in complex natural environments. We conducted an observational metagenomic study of the dynamics of interaction betweenGordoniaand their phages using a three-year data series of samples collected from a full-scale wastewater treatment plant. The aim was to obtain a comprehensive picture of the coevolution dynamics in naturally evolving populations at relatively high time resolution. Co-evolution was followed by monitoring changes over time in the CRISPR loci ofGordoniametagenome-assembled genome, and reciprocal changes in the viral genome. Genome-wide analysis indicated low strain variability ofGordonia, and almost clonal conservation of the trailer-end of the CRISPR loci. Incorporation of newer spacers gave rise to multiple coexisting bacterial populations. A host population containing a CRISPR array variant, which did not contain spacers against the coexisting phages, accounted for more than half of the total host abundance in the majority of samples. Phages genome co-evolved by introducing directional changes, with no preference for mutations within the protospacer and PAM regions. Metagenomic reconstruction of time-resolved variants of host and virus genomes revealed how selection operates at the population level. In activated sludge, it differed from the arms-race observed in nutrient rich media and resembled the fluctuating selection dynamics observed in natural environments.
Multi-parametric analysis of 58 SYNGAP1 variants reveal impacts on GTPase signaling, localization and protein stability
Authors: Meili Fabian , Wei William J. , Sin Wun-Chey , Dascalu Iulia , Callaghan Daniel B. , Rogic Sanja , Meyers Warren M. , Pavlidis Paul , Haas Kurt
Published: April 2020

SYNGAP1 is a Ras and Rap GTPase with important roles in regulating excitatory synaptic plasticity. While manySYNGAP1missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia and autism spectrum disorder (ASD), there are many variants of unknown significance (VUS). In this report, we characterize 58 variants in nine assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure the impact of variants on pERK, pGSK3β and pCREB and high-content imaging to examine their subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wildtype (WT)-like in their ability to regulate pERK and pGSK3β, while all variants retain at least partial ability to regulate pCREB. Interestingly, our assays reveal that a high percentage of variants located within the disordered domain of unknown function that makes up the C-terminal half of SYNGAP1 exhibited LoF, compared to the more well studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction only for two missense variants both located within the catalytic domain. Using high-content imaging, we find variants with nuclear enrichment/exclusion and aberrant nuclear speckle localization. These variants are primarily located within the C2 domain known to mediate membrane lipid interactions. We find that mislocalization is distinct from altered catalytic activity, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence pathogenicity classification.
A Cytoskeletal Function for PBRM1 Reading Methylated Microtubules
Authors: Karki Menuka , Jangid Rahul K. , Seervai Riyad N.H. , Bertocchio Jean-Philippe , Hotta Takashi , Msaouel Pavlos , Jung Sung Yun , Grimm Sandra L. , Coarfa Cristian , Weissman Bernard E. , Ohi Ryoma , Verhey Kristen J. , Hodges H. Courtney , Dere Ruhee , Park In-Young , Rathmell W. Kimryn , Walker Cheryl L. , Tripathi Durga N.
Published: April 2020

The chromatin modifier SETD2 was recently shown to be a dual-function methyltransferase that “writes” methyl marks on both chromatin and the mitotic spindle, revealing α-tubulin methylation as a new posttranslational modification of microtubules. Here, we report the first cytoskeletal “reader” for this SETD2 methyl mark: the polybromo protein PBRM1. We found PBRM1 directly binds the α-Tub-K40me3 mark on tubulin, and localizes to the mitotic spindle and spindle pole during cell division. PBRM1 can assemble a PBAF complex in the absence of chromatin as revealed by mass spectrometry, and can recruit other PBAF complex components including SMARCA4 and ARID2 to α-tubulin. In addition to PBRM1, other PBAF components were also localized to the mitotic spindle and spindle pole. This PBAF localization was dependent on recruitment to microtubules by PBRM1, and loss of spindle-associated PBRM1/PBAF led to genomic instability as assessed by increased formation of micronuclei. These data reveal a previously unknown function for PBRM1 beyond its role remodeling chromatin, and expand the repertoire of chromatin remodelers involved in writing and reading methyl marks on the cytoskeleton. The results of this study lay the foundation for a new paradigm for the epigenetic machinery as chromatocytoskeletal modifiers, with coordinated nuclear and cytoskeletal functions.
protein-sol pKa: prediction of electrostatic frustration, with application to coronaviruses
Authors: Hebditch Max , Warwicker Jim
Published: April 2020

Evolution couples differences in ambient pH to biological function through protonatable groups, in particular those that switch from buried to exposed and alter protonation state in doing so. We present a tool focusing on structure-based discovery and display of these groups. Since prediction of buried group pKas is computationally intensive, solvent accessibility of ionisable groups is displayed, from which the user can iteratively select pKa calculation centers. Results are color-coded, with emphasis on buried groups. Utility is demonstrated with coronaviruses, which exhibit variable dependence on the acidic pH of the endocytotic pathway. After benchmarking with variants of murine hepatitis virus, a pair of conserved histidine residues are identified that are predicted to be electrostatically frustrated at acidic pH in a common structural core of pre- and post-fusion coronavirus spike proteins. We suggest that an intermediate expanded conformation at endosomal pH could relax the frustration, allowing histidine protonation, and facilitating conformational conversion. This tool is available athttp://www.protein-sol.manchester.ac.uk/pka/.
Localized mRNA translation mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis
Authors: Fingerhut Jaclyn M , Yamashita Yukiko M
Published: April 2020

Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g. Plasmodium microgametocytes and human and Drosophila sperm), very little is known about cytoplasmic cilia assembly. Here we show that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the localized translation of these axonemal dyneins and their incorporation into the axoneme directly from the cytoplasm. We found that this RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. We propose that cytoplasmic cilia require the local translation of key protein constituents such that these proteins are incorporated efficiently into the axoneme.

Author Summary

Cytoplasmic cilia, which are found in human and Drosophila sperm, are unique in that the axoneme is exposed to the cytoplasm. The authors show that a novel RNP granule containing axonemal dynein mRNAs facilitates localized translation of these axonemal proteins, facilitating cytoplasmic cilia formation.
GMP-grade neural progenitor derivation and differentiation from clinical-grade human embryonic stem cells
Authors: Vitillo Loriana , Durance Catherine , Hewitt Zoe , Moore Harry , Smith Austin , Vallier Ludovic
Published: April 2020

A major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as Good Manufacturing Practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited. Here we present a new GMP-compliant protocol to derive long-term neuroepithelial stem cell progenitors (lt-NES), which are multipotent, bankable, and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells, deposited in the UK Stem Cell Bank, from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types. Overall, we report the feasibility of derivation and differentiation of clinical grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.
TBX3 acts as tissue-specific component of the Wnt/β-catenin enhanceosome
Authors: Zimmerli Dario , Borrelli Costanza , Jauregi-Miguel Amaia , Söderholm Simon , Brütsch Salome , Doumpas Nikolaos , Reichmuth Jan , Murphy-Seiler Fabienne , Aguet Michel , Basler Konrad , Moor Andreas E. , Cantù Claudio
Published: April 2020

BCL9 and PYGO are β-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signalling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of β-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the β-catenin transcriptional complex. In developing forelimbs, TBX3 and BCL9 co-occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations inBcl9affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a β-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as a new, context-dependent component of the Wnt/β-catenin-dependent enhanceosome.
The number of growing microtubules and nucleus-nucleus interactions uniquely regulate nuclear movement inDrosophilamuscle
Authors: Collins Mary Ann , Coon L. Alexis , Thomas Riya , Mandigo Torrey R. , Wynn Elizabeth , Folker Eric S.
Published: April 2020

Nuclear movement is a fundamental process of eukaryotic cell biology. Skeletal muscle presents an intriguing model to study nuclear movement because its development requires the precise positioning of multiple nuclei within a single cytoplasm. Furthermore, there is a high correlation between aberrant nuclear positioning and poor muscle function. Although many genes that regulate nuclear movement have been identified, the mechanisms by which these genes act is not known. UsingDrosophila melanogastermuscle development as a model system, and a combination of live-embryo microscopy and laser ablation of nuclei, we have found that phenotypically similar mutants are based in different molecular disruptions. Specifically, ensconsin (DrosophilaMAP7) regulates the number of growing microtubules that are used to move nuclei whereas bocksbeutel (Drosophilaemerin) and klarsicht (DrosophilaKASH-protein regulate interactions between nuclei.
Radiochromic film dosimetry in synchrotron radiation breast computed tomography: a phantom study
Authors: Mettivier Giovanni , Masi Marica , Arfelli Fulvia , Brombal Luca , Delogu Pasquale , Di Lillo Francesca , Donato Sandro , Fedon Christian , Golosio Bruno , Oliva Piernicola , Rigon Luigi , Sarno Antonio , Taibi Angelo , Russo Paolo
Published: April 2020

This study relates to the INFN project SYRMA-3D for in vivo phase-contrast breast computed tomography using the SYRMEP synchrotron radiation beamline at the ELETTRA facility in Trieste, Italy. This peculiar imaging technique uses a novel dosimetric approach with respect to the standard clinical procedure. In this study, optimization of the acquisition procedure was evaluated in terms of dose delivered to the breast. An offline dose monitoring method was also investigated using radiochromic film dosimetry. Various irradiation geometries have been investigated for scanning the prone patient's pendant breast, simulated by a 14 cm-diameter polymethylmethacrylate cylindrical phantom containing pieces of calibrated radiochromic film type XR-QA2. Films were inserted mid-plane in the phantom, as well as wrapped around its external surface, and irradiated at 38 keV, with an air kerma value that would produce an estimated mean glandular dose of 5 mGy for a 14 cm-diameter 50% glandular breast. Axial scans were performed over a full rotation or over 180°. The results point out that a scheme adopting a stepped rotation irradiation represents the best geometry to optimize the dose distribution to the breast. The feasibility of using a piece of calibrated radiochromic film wrapped around a suitable holder around the breast to monitor the scan dose offline is demonstrated.
A decagonal quasicrystal with rhombic and hexagonal tiles decorated with icosahedral structural units
Authors: Wang W. Z. , Zhou X. Z. , Yang Z. Q. , Qi Y. , Ye H. Q.
Published: April 2020

The structure of a decagonal quasicrystal in the Zn58Mg40Y2 (at.%) alloy was studied using electron diffraction and atomic resolution Z-contrast imaging techniques. This stable Frank–Kasper Zn–Mg–Y decagonal quasicrystal has an atomic structure which can be modeled with a rhombic/hexagonal tiling decorated with icosahedral units at each vertex. No perfect decagonal clusters were observed in the Zn–Mg–Y decagonal quasicrystal, which differs from the Zn–Mg–Dy decagonal crystal with the same space group P10/mmm. Y atoms occupy the center of `dented decagon' motifs consisting of three fat rhombic and two flattened hexagonal tiles. About 75% of fat rhombic tiles are arranged in groups of five forming star motifs, while the others connect with each other in a `zigzag' configuration. This decagonal quasicrystal has a composition of Zn68.3Mg29.1Y2.6 (at.%) with a valence electron concentration (e/a) of about 2.03, which is in accord with the Hume–Rothery criterion for the formation of the Zn-based quasicrystal phase (e/a = 2.0–2.15).
Investigation and multi-scale optimization design of woven composite cut-out structures
Authors: Nguyen Duc Hai , Wang Hu , Ye Fan , Hu Wei
Published: April 2020

Purpose

The purpose of this paper is to investigate the mechanical properties’ behaviors of woven composite cut-out structures with specific parameters. Because of the complexity of micro-scale and meso-scale structure, it is difficult to accurately predict the mechanical material behavior of woven composites. Numerical simulations are increasingly necessary for the design and optimization of test procedures for composite structures made by the woven composite. The results of the proposed method are well satisfied with the results obtained from the experiment and other studies. Moreover, parametric studies on different plates based on the stacking sequences are investigated.

Design/methodology/approach

A multi-scale modeling approach is suggested. Back-propagation neural networks (BPNN), radial basis function (RBF) and least square support vector regression are integrated with efficient global optimization (EGO) to reduce the weight of assigned structure. Optimization results are verified by finite element analysis.

Findings

Compared with other similar studies, the advantage of the suggested strategy uses homogenized properties behaviors with more complex analysis of woven composite structures. According to investigation results, it can be found that 450/−450 ply-orientation is the best buckling load value for all the cut-out shape requirements. According to the optimal results, the BPNN-EGO is the best candidate for the EGO to optimize the woven composite structures.

Originality/value

A multi-scale approach is used to investigate the mechanical properties of a complex woven composite material architecture. Buckling of different cut-out shapes with the same area is surveyed. According to investigation, 45°/−45° ply-orientation is the best for all cut-out shapes. Different surrogate models are integrated in EGO for optimization. The BPNN surrogate model is the best choice for EGO to optimization difficult problems of woven composite materials.
Does political instability influence dividend payout policy: evidence from Tunisian Stock Exchange?
Authors: Loukil Nadia
Published: April 2020

Purpose

The purpose of this study tests whether political instability influence financial decision-making behavior of Tunisian-listed firms, in particular dividend payout policy.

Design/methodology/approach

This paper uses dividend payout decisions announced over the period 2008–2015 by nonfinancial firms listed on the Tunisian Stock Exchange. A logistic regression is applied to analyze the relationship between political instability and dividend payout decision “changes. These latter are: past non-payers” dividend initiation, past payers' dividend termination, dividend payout “increasing and dividend payout” decreasing. Political instability variables used are as follows: number of changes in government head and dummy variables indicating the changes of ruling party and election year.

Findings

This study shows that government head changes are positively related to dividend initiation decisions while changes in ruling party are negatively related to termination dividend decisions except for family controlled ones. These firms are more likely to stop dividend on period of ruling party changes. Moreover, firms become unwilling to increase dividend payment on the period of political instability (changes in ruling party and government head and elections) and become willing to decrease dividend payment only when the government head changes.

Practical implications

The empirical findings contribute to the current debate on the signaling power of dividend policy in emerging market where raising equity capital is difficult and controlling shareholders prefer reinvest benefit to pay dividends. In addition, this study has important implications for regulators and governments struggling to design policies to improve investors' confidence and boost market activity. Indeed, investors may use corporate payout as a signal for better governance.

Originality/value

To the author' best knowledge, this paper is the first to investigate and to compare the effect of three political instability sources; government head changes, changes in ruling party and elections, on dividend payout decision changes. This paper provides evidence that firms facing political unstable environment seek to achieve two goals when they make dividend policy: reducing financial distress probability and attracting minority owners.
HRM practices and innovation: an empirical systematic review
Authors: Easa Nasser Fathi , Orra Haitham El
Published: April 2020

Purpose

The relationship between human resource management practices (HRMP) and innovation has been described as a black box, where a lot still needs to be investigated. Thus, the aim of this paper is to investigate the nature of the link that exists between HRMP and innovation in both public and private organizations. To do so, theoretical underpinnings and existence of a mediating or a moderating mechanism is inspected.

Design/methodology/approach

Based on an empirical systematic review of research conducted between 2010 and 2018, content analysis has been conducted for 31 peer-reviewed articles in the English language.

Findings

Inspecting the nature of relations existed in the chosen articles, interesting findings are addressed relative to the nature of the human resource management systems (HRMS) used, practices encompassed and their different utility. HRMS has been shown to be associated with product innovation yet more evidence is needed for supporting process innovation.

Practical implications

The HRMS/HRMP and innovation relationship is inspected, important practices that would guide managers to induce innovation are highlighted. Usage of multiple HRMS and contingency in constructing such systems is indicated.

Originality/value

Contribution to comprehend the black box and areas for future research has been offered.
Discomfort and organizational change as a part of becoming a world-class university
Authors: Nadia Fiona Niska Dinda , Sukoco Badri Munir , Susanto Ely , Sridadi Ahmad Rizki , Nasution Reza Ashari
Published: April 2020

Purpose

This study examined organizational change in universities as it relates to discomfort among the organization's members.

Design/methodology/approach

Using the critical incident technique (CIT), data was collected from the informants in an Indonesian public university that had been mandated by the government to enter the top 500 world university ranking. This would make it a “World-Class” university.

Findings

The findings describe the causes, courses and consequences of the discomfort felt in response to the organizational change in the university context. The causes of discomfort were categorized as a fear of loss, organizational culture, systems and policies, work overload and a lack of resources. Discomfort can manifest through negative affective, cognition and behavioral tendencies. Meanwhile, the consequences result in active and passive participation in the process of the organizational change itself.

Originality/value

Discomfort with organizational change is a new variable that has rarely been explored, thus it requires testing and validation using different methods and contexts, as offered by this study. We have also shown that in the initial stage of organizational change (unfreezing), discomfort will always emerge that must be immediately managed in order not to trigger resistance to change. Furthermore, this study exhibits the use of the critical incident technique in the context of organizational change. Finally, we offer comprehensive views by exhibiting the causes, the reactions shown and the consequences of discomfort with the change.