bluuebunny/crossref_metadata_2025_split huggingface dataset:
Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS-CoV-2 variants
Authors: Wang Qiong , Ye Sheng-Bao , Zhou Zhi-Jian , Li Jin-Yan , Lv Ji-Zhou , Hu Bodan , Yuan Shuofeng , Qiu Ye , Ge Xing-Yi 
Published: April 2022

AbstractIncreasing evidence supports inter-species transmission of SARS-CoV-2 variants from human to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, in order to evaluate the impact of S mutations, we constructed 20 Hela cell lines stably expressing ACE2 orthologs from different animals, and prepared 27 pseudotyped SARS-CoV-2 carrying different spike mutants, among which 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.135), Lambda (B.1.429) and Mu (B.1.525). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammals ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations.
The ADAM17 sheddase complex regulator iTAP modulates inflammation, epithelial repair, and tumor growth
Authors: Badenes Marina , Burbridge Emma , Oikonomidi Ioanna , Amin Abdulbasit , de Carvalho Érika , Kosack Lindsay , Domingos Pedro , Faísca Pedro , Adrain Colin 
Published: April 2022

AbstractThe metalloprotease ADAM17 catalyzes the shedding of key signalling molecules from the cell surface, including the inflammatory cytokine TNF (tumour necrosis factor) and activating ligands of the EGFR (epidermal growth factor receptor). ADAM17 exists within an assemblage called the “sheddase complex” containing a rhomboid pseudoprotease (iRhom1 or iRhom2). iRhoms control multiple aspects of ADAM17 biology, including its vesicular trafficking, maturation from its precursor pro-form, activation on the cell surface and specificity for subsets of proteolytic targets. Previous studies from our laboratory and others identified the FERM domain-containing protein Frmd8/iTAP as an iRhom-binding protein. iTAP is required to maintain the cell surface stability of the sheddase complex, thereby preventing the precocious shunting of ADAM17 and iRhom2 to lysosomes and their consequent degradation. As pathophysiological role(s) of iTAP have not been addressed, here we sought to characterize the impact of loss of iTAP on ADAM17-associated phenotypes in mice. Our data show that iTAP KO mice exhibit defects in ADAM17 activity in inflammatory and intestinal epithelial barrier repair functions, but do not exhibit the collateral effects associated with global loss of ADAM17. Furthermore, we show that iTAP promotes cancer cell growth in a cell-autonomous manner, and by modulating the tumor microenvironment. Our work suggests that pharmacological intervention at the level of iTAP may be beneficial to target ADAM17 activity in specific compartments during chronic inflammatory diseases or cancer, avoiding the deleterious impact on vital functions associated with the widespread inhibition of ADAM17 in normal tissues.
A synthetic ancestral Kinesin-13 depolymerises microtubules faster than any natural depolymerising kinesin
Authors: Belsham Hannah R. , Alghamdi Hanan M. , Dave Nikita , Rathbone Alexandra J. , Wickstead Bill , Friel Claire T. 
Published: April 2022

AbstractThe activity of a kinesin is largely determined by the ~350 residue motor domain and this region alone is sufficient to classify a kinesin as a member of a particular family. The Kinesin-13 family are a group of microtubule depolymerising kinesins and are vital regulators of microtubule length. Members of this family are critical to spindle assembly and chromosome segregation in both mitotic and meiotic cell division and play crucial roles in cilium length control and neuronal development. To better understand the evolution of microtubule depolymerisation activity in the Kinesin-13 family, we created a synthetic ancestral Kinesin-13 motor domain. This phylogenetically-inferred ancestral motor domain is the sequence predicted to have existed in the common ancestor of the Kinesin-13 family. Here we show that the ancestral Kinesin-13 motor depolymerises stabilised microtubules faster than any previously tested depolymerase. This potent activity is more than an order of magnitude faster than the most highly studied Kinesin-13, MCAK and allows the ancestral Kinesin-13 to depolymerise doubly-stabilised microtubules that are unaffected by MCAK. These data suggest that the ancestor of the Kinesin-13 family was a ‘super depolymeriser’ and that members of the Kinesin-13 family have evolved away from this extreme depolymerising activity to provide more controlled microtubule depolymerisation activity in extant cells.
Coarsening dynamics can explain meiotic crossover patterning in both the presence and absence of the synaptonemal complex
Authors: Fozard John A , Morgan Chris , Howard Martin 
Published: April 2022

AbstractThe shuffling of genetic material facilitated by meiotic crossovers is a critical driver of genetic variation. Therefore, the number and positions of crossover events must be carefully controlled. InArabidopsis, an obligate crossover and repression of nearby crossovers on each chromosome pair are abolished in mutants that lack the synaptonemal complex (SC), a conserved protein scaffold. We use mathematical modelling and quantitative super-resolution microscopy to explore and mechanistically explain meiotic crossover pattering inArabidopsislines with full, incomplete or abolished synapsis. Forzyp1mutants, which lack an SC, we develop a coarsening model in which crossover precursors globally compete for a limited pool of the pro-crossover factor HEI10, with dynamic HEI10 exchange mediated through the nucleoplasm. We demonstrate that this model is capable of quantitatively reproducing and predictingzyp1experimental crossover patterning and HEI10 foci intensity data. Additionally, we find that a model combining both SC- and nucleoplasm-mediated coarsening can explain crossover patterning in wild-typeArabidopsisand inpch2mutants, which display partial synapsis. Together, our results reveal that regulation of crossover patterning in wild-typeArabidopsisand SC defective mutants likely act through the same underlying coarsening mechanism, differing only in the spatial compartments through which the pro-crossover factor diffuses.
A diet-independent zebrafish model for NAFLD recapitulates patient lipid profiles and offers a system for small molecule screening
Authors: Singh Manoj K , Yadav Rohit , Bhaskar Akash Kumar , Sengupta Shantanu , Sachidanandan Chetana 
Published: April 2022

AbstractNon-alcoholic Fatty Liver Disease (NAFLD) or pathological hepatic lipid overload, is considered to affect obese individuals. However, NAFLD in lean individuals is prevalent, especially in South Asian population. The pathophysiology of lean NAFLD is not well understood and most animal models of NAFLD use the high-fat diet paradigm. To bridge this gap, we have developed a diet-independent model of NAFLD in zebrafish. We have previously showed that chronic systemic inflammation causes metabolic changes in the liver leading to hepatic fat accumulation in an IL6 overexpressing (IL6-OE) zebrafish model. In the present study, we compared the hepatic lipid composition of adult IL6-OE zebrafish to the controls and found an accumulation of saturated triacylglycerols and a reduction in the unsaturated triacylglycerol species reminiscent of NAFLD patients. Zebrafish is an ideal system for chemical genetic screens. We tested whether the hepatic lipid accumulation in the IL6-OE is responsive to chemical treatment. We found that PPAR-gamma agonist Rosiglitazone, known to reduce lipid overload in the high fat diet models of NAFLD, could ameliorate the fatty liver phenotype of the IL6-OE fish. Rosiglitazone treatment reduced the accumulation of saturated lipids and showed a concomitant increase in unsaturated TAG species in our inflammation-induced NAFLD model. Our observations suggest that the IL6-OE model can be effective for small molecule screening to identify compounds that can reverse hepatic lipid accumulation, especially relevant to lean NAFLD.
INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors
Authors: Hör Jens , Jung Jakob , Đurica-Mitić Svetlana , Barquist Lars , Vogel Jörg 
Published: April 2022

ABSTRACTRibosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has limitations. These include difficulties with detection of low abundance transcripts and analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developedin vitroRibo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. UsingEscherichia colias an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.
Separation of phosphatase and kinase activity within the Bub complex is required for proper mitosis
Authors: Wang Lei , Kruse Thomas , López-Méndez Blanca , Zhang Yuqing , Song Chunlin , Zhu Lei , Li Bing , Fang Jing , Lu Zhimin , Nilsson Jakob , Zhang Gang 
Published: April 2022

ABSTRACTThe Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogues with Bub1 being a kinase while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can supports chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation resulting in reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.
Autophagy Increases Occludin Levels to Enhance Intestinal Paracellular Tight Junction Barrier
Authors: Saha Kushal , Ganapathy Ashwinkumar Subramenium , Wang Alexandria , Morris Nathan Michael , Suchanec Eric , Yochum Gregory , Koltun Walter , Ding Wei , Nighot Meghali , Ma Thomas , Nighot Prashant 
Published: April 2022

AbstractBackground and AimFunctional loss of paracellular tight junction (TJ) barrier of the gut epithelium and mutations in autophagy genes are factors potentiating inflammatory bowel disease (IBD). Previously we showed the role of autophagy in enhancing the TJ barrier via claudin-2 degradation, however, its role in the regulation of the barrier-forming protein occludin remains unknown. Here, we investigate the role of autophagy in the regulation of occludin and its role in inflammation-mediated TJ barrier loss.MethodsPharmacological and genetic tools were used to study the effect of autophagy on occludin levels and localization, and the role of the MAPK pathway.ResultsAutophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in different epithelial cells. Starvation enriched membrane occludin levels and reduced paracellular inulin flux in Caco-2 cells. Starvation-induced TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier enhancing effect. Autophagy inhibited the constitutive degradation of occludin and protected against inflammation-induced TJ barrier loss. Starvation-induced TJ barrier enhancement was prevented by inhibition of autophagy. Autophagy enhanced the phosphorylation of ERK-1/2. Inhibition of these kinases in Caco-2 cells and human intestinal mucosa inhibited the protective effects of autophagy. In-vivo, autophagy induction by rapamycin increased occludin levels in mouse intestines and protected against LPS and TNF-α-induced TJ barrier loss. Additionally, acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ-permeability and exacerbating the effect of DSS-induced colitis.ConclusionOur data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 MAPK-dependent mechanism.Graphical abstract
Condensation of LINE-1 is required for retrotransposition
Authors: Sil Srinjoy , Boeke Jef D , Holt Liam J 
Published: April 2022

AbstractLINE-1 (L1) is the only autonomously active retrotransposon in the human genome, and accounts for 17% of the human genome. The L1 mRNA encodes two proteins, ORF1p and ORF2p. ORF1p is a homotrimeric RNA-binding protein that plays a critical role in assembling functional L1 ribonucleoprotein (RNP) complexes. Here we show that condensation of ORF1p is required for L1 retrotransposition. Using a combination of biochemical reconstitution and live-cell imaging, we demonstrate that RNA binding, electrostatic interactions, and trimer conformational dynamics together tune the properties of ORF1p assemblies to allow for efficient L1 condensate formation in cells. Furthermore, we directly relate the dynamics of ORF1p assembly to the ability to complete the entire retrotransposon life-cycle. Mutations that prevented ORF1 condensation led to loss of retrotransposition activity, while orthogonal restoration of coiled-coil conformational flexibility rescued both condensation and retrotransposition. Based on these observations, we propose that ORF1p oligomerization on L1 RNA drives the formation of a dynamic L1 condensate that is essential for retrotransposition.
Using soybean historical field trial data to study genotype by environment variation and identify mega-environments with the integration of genetic and non-genetic factors
Authors: Krause Matheus D , Dias Kaio O G , Singh Asheesh K , Beavis William D 
Published: April 2022

1AbstractSoybean (Glycine max(L.) Merr.) provides plant-based protein for global food production and is extensively bred to create cultivars with greater productivity in distinct environments. Plant breeders evaluate new soybean genotypes using multi-environment trials (MET). The application of MET assumes that trial locations provide representative environmental conditions that cultivars are likely to encounter when grown by farmers. In addition, MET are important to depict the patterns of genotype by environment interactions (GEI). To evaluate GEI for soybean seed yield and identify mega-environments (ME), a retrospective analysis of 39,006 data points from experimental soybean genotypes evaluated in preliminary and uniform field trials conducted by public plant breeders from 1989-2019 was considered. ME were identified from phenotypic information from the annual trials, geographic, soil, and meteorological records at the trial locations. Results indicate that yield variation was mostly explained by location and location by year interactions. The static portion of the GEI represented 26.30% of the total yield variance. Estimates of variance components derived from linear mixed models demonstrated that the phenotypic variation due to genotype by location interaction effects was greater than genotype by year interaction effects. A trend analysis indicated a two-fold increase in the genotypic variance between 1989-1995 and 1996-2019. Furthermore, the heterogeneous estimates of genotypic, genotype by location, genotype by year, and genotype by location by year variances, were encapsulated by distinct probability distributions. The observed target population of environments can be divided into at least two and at most three ME, thereby suggesting improvements in the response to selection can be achieved when selecting directly for clustered (i.e., regions, ME) versus selecting across regions. Clusters obtained using phenotypic data, latitude, and soil variables plus elevation, were the most effective. In addition, we published the R package SoyURT which contains the data sets used in this work.2HighlightsMega-environments can be identified with phenotypic, geographic, and meteorological data.Reliable estimates of variances can be obtained with proper analyses of historical data.Genotype by location was more important than genotype by year variation for seed yield.The trend in genotype by environment variances was captured in probability distributions.
Highly Parallelized, Multicolor Optogenetic Recordings of Cellular Activity for Therapeutic Discovery Applications in Ion Channels and Disease-associated Excitable Cells
Authors: Borja Gabriel B , Zhang Hongkang , Harwood Benjamin N , Jacques Jane , Grooms Jennifer , Chantre Romina , Zhang Dawei , Barnett Adam , Werley Christopher A , Lu Yang , Nagle Steven , McManus Owen B , Dempsey Graham T 
Published: April 2022

AbstractOptogenetic assays provide a flexible, scalable and information rich approach to probe compound effects for ion channel drug targets in both heterologous expression systems and associated disease relevant cell types. Despite the potential utility and growing adoption of optogenetics, there remains a critical need for compatible platform technologies with the speed, sensitivity, and throughput to enable their application to broader drug screening applications. To address this challenge, we developed the Swarm™, a custom designed optical instrument for highly parallelized, multicolor measurements in excitable cells, simultaneously recording changes in voltage and calcium activities at high temporal resolution under optical stimulation. The compact design featuring high power LEDs, large numerical aperture optics, and fast photodiode detection enables all-optical individual well readout of 24-wells simultaneously from multi-well plates while maintaining sufficient temporal resolution to probe millisecond response dynamics. The Swarm delivers variable intensity blue-light optogenetic stimulation to enable membrane depolarization and red or lime-light excitation to enable fluorescence detection of the resulting changes in membrane potential or calcium levels, respectively. The Swarm can screen ~10,000 wells / day in 384-well format, probing complex pharmacological interactions via a wide array of stimulation protocols. To evaluate the Swarm screening system, we optimized a series of heterologous optogenetic spiking HEK293 cell assays for several voltage-gated sodium channel subtypes including Nav1.2, Nav1.5, and Nav1.7. The Swarm was able to record pseudo-action potentials stably across all 24 objectives and provided pharmacological characterization of diverse sodium channel blockers. We performed a Nav1.7 screen of 200,000 small molecules in a 384-well plate format with all 560 plates reaching a Z’≥0.5. As a demonstration of the versatility of the Swarm, we also developed an assay measuring cardiac action potential and calcium waveform properties simultaneously under paced conditions using human induced pluripotent stem (iPS) cell-derived cardiomyocytes as an additional counter screen for cardiac toxicity. In summary, the Swarm is a novel high-throughput all-optical system capable of collecting information-dense data from optogenetic assays in both heterologous and iPS cell-derived models, which can be leveraged to drive diverse therapeutic discovery programs for nervous system disorders and other disease areas involving excitable cells.
Olfactory receptor neurons generate multiple response motifs, increasing coding space dimensionality
Authors: Kim Brian , Haney Seth , Millan Ana P. , Joshi Shruti , Aldworth Zane , Rulkov Nikolai , Kim Alexander T. , Bazhenov Maxim , Stopfer Mark 
Published: April 2022

ABSTRACTOdorants binding to olfactory receptor neurons (ORNs) trigger bursts of action potentials, providing the brain with its only experience of the olfactory environment. Our recordings made in vivo from locust ORNs showed odor-elicited firing patterns comprise four distinct response motifs, each defined by a reliable temporal profile. Different odorants could elicit different response motifs from a given ORN, a property we term motif switching. Further, each motif undergoes its own form of sensory adaptation when activated by repeated plume-like odor pulses. A computational model constrained by our recordings revealed that organizing responses into multiple motifs provides substantial benefits for classifying odors and processing complex odor plumes: each motif contributes uniquely to encode the plume’s composition and structure. Multiple motifs and motif switching further improve odor classification by expanding coding dimensionality. Our model demonstrated these response features could provide benefits for olfactory navigation, including determining the distance to an odor source.