The Shigella type III effector protein OspB is a cysteine protease The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and dysenteric disease. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes.

ImportanceShigella spp. are important human pathogens and one of the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS), and definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB has been shown to contribute to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad.
Invasive grass dominance over native forbs is linked to shifts in the bacterial rhizosphere microbiome Rhizosphere microbiomes have received growing attention in recent years for their role in plant health, stress tolerance, soil nutrition, and invasion. Still, relatively little is known about how these microbial communities are altered under plant competition, and even less about whether these shifts are tied to competitive outcomes between native and invasive plants. We investigated the structure and diversity of rhizosphere bacterial and fungal microbiomes of native annual forbs and invasive annual grasses individually and in competition using high-throughput amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS region. We assessed how differentially abundant microbial families correlate to plant biomass under competition We find that bacterial diversity and structure differ between native forbs and invasive grasses, but fungal diversity and structure do not. Further, bacterial community structures under competition are distinct from individual bacterial community structures. We also identified five bacterial families that varied in normalized abundance between treatments and that were correlated with plant biomass under competition. We speculate that invasive grass dominance over these natives may be partially due to effects on the rhizosphere community, with changes in specific bacterial families potentially benefiting invaders at the expense of natives.
A novel Fiji/ImageJ plugin for the rapid analysis of blebbing cells When confined, cells have recently been shown to undergo a phenotypic switch to what has been termed, fast amoeboid (leader bleb-based) migration. However, as this is a nascent area of research, few tools are available for the rapid analysis of cell behavior. Here, we demonstrate that a novel Fiji/ImageJ-based plugin, Analyze_Blebs, can be used to quickly obtain cell migration parameters and morphometrics from time lapse images. As validation, we show that Analyze_Blebs can detect significant differences in cell migration and morphometrics, such as the largest bleb size, upon introducing different live markers of F-actin, including F-tractin and LifeAct tagged with green and red fluorescent proteins. We also demonstrate, using flow cytometry, that live markers increase total levels of F-actin. Furthermore, that F-tractin increases cell stiffness, which was found to correlate with a decrease in migration, thus reaffirming the importance of cell mechanics as a determinant of Leader Bleb-Based Migration (LBBM).

Highlight summaryA new plugin, Analyze_Blebs, enables the rapid analysis of cell migration and morphometrics of fast amoeboid cells. Morphometrics combined with cell stiffness are found to be predictive of confined, fast amoeboid (leader bleb-based), migration. Environment dependent benefits of inter-individual variation in honey bee recruitment Inter-individual differences in behaviour within the members of a social group can affect the groups productivity. In eusocial insects, individual differences amongst workers in a colony play a central role division of labour and task allocation. Extensive empirical and theoretical work has highlighted variation in response thresholds as a proximate mechanism underlying individual behavioural differences and hence division of labour. However, other response parameters, like response probability and intensity, can affect these differences. In this study, we first extended a previously published agent-based model on honey bee foraging to understand the relative importance of response (dance) probability and response (dance) intensity in the task of recruitment. Comparing variation obtained from the simulations with previously published empirical data, we found that response intensity plays a more important role than probability in producing consistent inter-individual differences in recruitment behaviour. We then explored the benefits provided by this individual variation in recruitment behaviour to the colonys collective foraging effort under different environmental conditions. Our results revealed that individual variation leads to a greater energetic yield per forager, but only when food is abundant. Our study highlights the need to consider all response parameters while studying division of labour and adds to the growing body of evidence linking individual variation in behavioural responses to the success of social groups. Lymph node swelling combined with temporary effector T cell retention aids T cell response in a model of adaptive immunity Swelling of lymph nodes is commonly observed during the adaptive immune response, yet the impact on T cell trafficking and subsequent immune response is not well known. To better understand the effect of macro-scale alterations, we developed an agent-based model of the lymph node para-cortex, describing T cell trafficking and response to antigen-presenting dendritic cells alongside swelling-induced changes in T cell recruitment and egress, and regulation of expression of egress-modulating T cell receptor Sphingosine-1-phosphate receptor-1. Analysis of effector T cell response under varying swelling conditions showed that swelling consistently aided T cell activation. However, subsequent effector CD8+ T cell production could be reduced if swelling occurred too early in the T cell proliferative phase or if T cell cognate frequency was low due to increased opportunity for T cell exit. Temporarily extending retention of newly differentiated effector T cells, mediated by Sphingosine-1-phosphate receptor-1 expression, mitigated any negative effects of swelling by allowing facilitation of activation to outweigh increased access to exit areas. These results suggest targeting temporary effector T cell retention and egress associated with swelling may offer new ways to modulate effector TC responses in, for example, immuno-suppressed patients and optimisation of vaccine design. A novel nongenetic murine model of hyperglycemia and hyperlipidemia-associated aggravated atherosclerosis ObjectiveAtherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s).

Methods and ResultsWild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less -SMA-positive area) and had more inflammation (increased plasma IL-1{beta} level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks.

ConclusionWe established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice. Lipid membranes modulate the activity of RNA through sequence-dependent interactions RNA is a ubiquitous biomolecule that can serve as both catalyst and information carrier. Understanding how RNA bioactivity is controlled is crucial for elucidating its physiological roles and potential applications in synthetic biology. Here we show that lipid membranes can act as RNA organization platforms, introducing a novel mechanism for ribo-regulation. The activity of R3C ribozyme can be modified by the presence of lipid membranes, with direct RNA-lipid interactions dependent on RNA nucleotide content, base pairing and length. In particular, the presence of guanine in short RNAs is crucial for RNA-lipid interactions, and G-quadruplex formation further promotes lipid binding. Lastly, by artificially modifying the R3C substrate sequence to enhance membrane binding we generated a lipid-sensitive ribozyme reaction with riboswitch-like behavior. These findings introduce RNA-lipid interactions as a tool for developing synthetic riboswitches and novel RNA-based lipid biosensors, and bear significant implications for RNA World scenarios for the origin of life. Localization of signaling receptors maximizes cellular informationacquisition in spatially-structured natural environments Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization maximizes information acquisition in simulated natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold. Broadly, our work shows how cells can maximize the fidelity of information transfer by adapting the spatial organization of signaling molecules to the spatial structure of the environment. Classification, Using Field Potential Images, of Pacemaker Micro-coordination in the Mouse Intestine The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals. Multivariate genome-wide association study on tissue-sensitive diffusion metrics identifies key molecular pathways for axonal growth, synaptogenesis, and astrocyte-mediated neuroinflammation The molecular determinants of tissue composition of the human brain remain largely unknown. Recent genome-wide association studies (GWAS) on this topic have had limited success due to methodological constraints. Here, we apply advanced whole-brain analyses on multi-shell diffusion imaging data and multivariate GWAS to two large scale imaging genetic datasets (UK Biobank and the Adolescent Brain Cognitive Development study) to identify and validate genetic association signals. We discovered 503 unique genetic loci that have impact on multiple regions of human brain. Among them, more than 79% were validated in either of two large-scale independent imaging datasets. Key molecular pathways involved in axonal growth, astrocyte-mediated neuroinflammation, and synaptogenesis during development were found to significantly impact the measured variations in tissue-specific imaging features. Our results shed new light on the biological determinants of brain tissue composition and their potential overlap with the genetic basis of neuropsychiatric disorders. Effects of phenotypic variation on consumer coexistence and prey community structure A popular idea in ecology is that trait variation among individuals from the same species may promote the coexistence of competing species. However, theoretical and empirical tests of this idea have yielded inconsistent findings. We manipulated intraspecific trait diversity in a ciliate competing with a nematode for bacterial prey in experimental microcosms. We found that intraspecific trait variation inverted the original competitive hierarchy to favor the consumer with variable traits, ultimately resulting in competitive exclusion. This competitive outcome was driven by foraging traits (size, speed, and directionality) that increased the ciliates fitness ratio and niche overlap with the nematode. The interplay between consumer trait variation and competition resulted in non-additive cascading effects - mediated through prey defense traits - on prey community assembly. Our results suggest that predicting consumer competitive population dynamics and the assembly of prey communities will require understanding the complexities of trait variation within consumer species. The evolution of stomatal traits along the trajectory towards C4 photosynthesis C4 photosynthesis optimizes plants carbon and water relations, allowing high photosynthetic rate with low stomatal conductance. Stomata have long been believed as a part of C4 syndrome. However, it remains unclear how stomata traits evolved along the path from C3 to C4. Stomatal patterning was examined in Flaveria genus, a model for studying C4 evolution. Comparative, transgenic and semi-in-vitro experiments were used to study molecular basis that underlies stomatal traits along C4 evolution. Novel results: the evolution from C3 to C4 species through intermediate species is accompanied by a stepwise rather than an abrupt change in the stomatal traits. The initial change occurs near Type II and dramatic change occurs at the C4-like species. On the road to C4, stomata become less in number but bigger in size and changes in stomatal density dominates changes in maximum stomatal conductance (gsmax). The reduction of FSTOMAGEN expression underlies altered gsmax between Flaveria species with different photosynthetic pathways and likely occurs in other C4 lineages. Our study provides insight into the pattern, mechanism and role of stomatal evolution along the road towards C4. This work highlights the stomatal traits in the current C4 evolutionary model and the co-evolution of photosynthetic pathway and stomata. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. TCF11 Has a Potent Tumor-Repressing Effect than Its Prototypic Nrf1α by Definition of both Similar yet Different Regulatory Profiles, with a Striking Disparity from Nrf2 Nrf1 and Nrf2, as two principal CNC-bZIP transcription factors, regulate similar but different targets involved in a variety of biological functions for maintaining cell homeostasis and organ integrity. Of note, the unique topobiological behavior of Nrf1 makes its functions more complicated than Nrf2, because it is allowed for alternatively transcribing and selectively splicing to yield multiple isoforms (e.g., TCF11, Nrf1). In order to gain a better understanding of their similarities and differences in distinct regulatory profiles, all four distinct cell models for stably expressing TCF11, TCF11{Delta}N, Nrf1 or Nrf2 have been herein established by an Flp-In T-REx-293 system and then identified by transcriptomic sequencing. Further analysis revealed that Nrf1 and TCF11 have similar yet different regulatory profiles, although both contribute basically to positive regulation of their co-targets, which are disparate from those regulated by Nrf2. Such disparity in those gene regulation by Nrf1 and Nrf2 was further corroborated by scrutinizing comprehensive functional annotation of their specific and/or common target genes. Conversely, the mutant TCF11{Delta}N, resulting from a deletion of the N-terminal amino acids 2-156 from TCF11, resembles Nrf2 with the largely consistent structure and function. Interestingly, our further experimental evidence demonstrates that TCF11 acts as a potent tumor-repressor relative to Nrf1, albeit both isoforms possess a congruous capability to prevent malignant growth of tumor and upregulate those genes critical for improving the survival of patients with hepatocellular carcinoma. First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI Eyeblink conditioning performance and brain-wide C-fos expression in male and female mice The functional and molecular sources of behavioral variability in mice are not fully understood. As a consequence, the predominant use of male mice has become a standard in animal research, under the assumption that males are less variable than females. Similarly, to homogenize genetic background, neuroscience studies have almost exclusively used the C57BL/6 (B6) strain. Here, we examined individual differences in performance in the context of associative learning. We performed delayed eyeblink conditioning while recording locomotor activity in mice from both sexes in two strains (B6 and B6CBAF1). Further, we used a C-FOS immunostaining approach to explore brain areas involved in eyeblink conditioning across subjects and correlate them with behavioral performance. We found that B6 male and female mice show comparable variability in this task and that females reach higher learning scores. We found a strong positive correlation across sexes between learning scores and voluntary locomotion. C-FOS immunostainings revealed positive correlations between C-FOS positive cell density and learning in the cerebellar cortex, as well as multiple previously unreported extra-cerebellar areas. We found consistent and comparable correlations in eyeblink performance and C-fos expression in B6 and B6CBAF1 females and males. Taken together, we show that differences in motor behavior and activity across brain areas correlate with learning scores during eyeblink conditioning across strains and sexes. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Sufu regulation of Hedgehog signaling in P19 cells is required for proper astrocyte differentiation Hedgehog signaling is essential for vertebrate development, however, less is known about the negative regulators that influence this pathway during the differentiation of cell fates. Using the mouse P19 embryonal carcinoma cell model, Suppressor of Fused (SUFU), a negative regulator of the Hedgehog pathway, was investigated during retinoic acid-induced neural differentiation. We found Hedgehog signaling was activated in the early phase of neural differentiation but was inactive during terminal differentiation of neurons and astrocytes. This early activation was required for neural differentiation however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of GLI, remained relatively unchanged during the differentiation process, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hedgehog target genes. Interestingly, these SUFU-deficient cells were unable to differentiate in the absence of retinoic acid, but when differentiated using retinoic acid, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout retinoic acid-induced differentiation and this phenotype was accompanied by the loss of GLI3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, and its normal regulation of GLI3 to maintain Hh signaling in an inactive state. Thirteen independent genetic loci associated with preserved processing speed in a study of cognitive resilience in 330,097 individuals in the UK Biobank Cognitive resilience is the ability to withstand the negative effects of stress on cognitive functioning and is important for maintaining quality of life while aging. UK Biobank (UKB) does not have direct measurements of the same cognitive phenotype at distal timepoints. Therefore, we used number of education years (EY) as a proxy phenotype for past cognitive performance. Current cognitive performance was determined based on processing speed. This approach captured an average time span of 40 years between past and current cognitive performance in 330,097 individuals. A confounding factor was that EY is highly polygenic and masked the genetics of resilience. To overcome this, we employed Genomics Structural Equation Modelling (GenomicSEM) to perform a GWAS-by-subtraction using two GWAS, one GWAS of EY and resilience and a second GWAS of EY but not resilience. Subtracting one from the other generated a GWAS of Resilience. Replication of this approach was shown using independent discovery and replication samples within UKB and the full UKB GWAS results show significant genetic correlation with a GWAS of cognitive change in the independent Health and Retirement Study (N=9,526; P=1.5x10-3). We found 13 independent genetic loci for Resilience. Functional analyses showed enrichment in several brain regions and involvement of specific cell types, including GABAergic neurons (P=6.59x10-8) and glutamatergic neurons (P=6.98x10-6) in the cortex. Gene-set analyses implicated the biological process "neuron differentiation" (P=9.7x10-7) and the cellular component "synaptic part" (P=2.14x10-6). Mendelian randomization analysis showed a causative effect of white matter volume on cognitive resilience. These results enhance neurobiological understanding of resilience. A Comparative Analysis of Novel Deep Learning and Ensemble Learning Models to Predict the Allergenicity of Food Proteins Traditional food allergen identification mainly relies on in vivo and in vitro experiments, which often needs a long period and high cost. The artificial intelligence (AI)-driven rapid food allergen identification method has solved the above mentioned two drawbacks and is becoming an efficient auxiliary tool. Aiming to overcome the limitations of lower accuracy of traditional machine learning models in predicting the allergenicity of food proteins, this work proposed to introduce deep learning model - transformer with self-attention mechanism, ensemble learning models (representative as Light Gradient Boosting Machine (LightGBM) eXtreme Gradient Boosting (XGBoost)) to solve the problem. In order to highlight the superiority of the proposed novel method, the study also selected various commonly used machine learning models as the baseline classifiers. The results of 5-fold cross-validation showed that the AUC of the deep model was the highest (0.9578), which was better than the ensemble learning and baseline algorithms. But the deep model need to be pre-trained, and the training cost is the highest. By comparing the characteristics of the transformer model and boosting models, it can be analyzed that, each model has its own advantage, which provides novel clues and inspiration for the rapid prediction of food allergens in the future. Targeting SARS-CoV-2 infection through CAR-T like bispecific T cell engagers incorporating ACE2 Despite advances in antibody treatments and vaccines, COVID-19 caused by SARS-CoV-2 infection remains a major health problem resulting in excessive morbidity and mortality and the emergence of new variants has reduced the effectiveness of current vaccines. Here, as a proof-of-concept we engineered primary CD8 T cells to express SARS-CoV-2 Spike protein-specific CARs, using extracellular region of ACE2, and demonstrated their highly specific and potent cytotoxicity towards Spike-expressing target cells. To improve on this concept as a potential therapeutic, we developed a bispecific T cell engager combining ACE2 with an anti-CD3 scFv (ACE2-Bite) to target infected cells and the virus. Similar to CAR-T cell approach, ACE2-Bite endowed cytotoxic cells to selectively kill Spike-expressing targets. Furthermore, ACE2-Bite neutralized the pseudoviruses of SARS-CoV, SARS-CoV-2 wild-type and variants including Delta and Omicron, as a decoy protein. Remarkably, ACE2-Bite molecule showed a higher binding and neutralization affinity to Delta and Omicron variants compared to SARS-CoV-2 wild-type Spike proteins, suggesting the potential of this approach as a variant-proof, therapeutic strategy for future SARS-CoV-2 variants, employing both humoral and cellular arms of the adaptive immune response. SERCA function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice. Chromatin-contact atlas reveals disorder-mediated protein interactions and moonlighting chromatin-associated RBPs RNA-binding proteins (RBPs) play diverse roles in regulating co-transcriptional RNA-processing and chromatin functions, but our knowledge of the repertoire of chromatin-associated RBPs (caRBPs) and their interactions with chromatin remains limited. Here, we developed SPACE (Silica Particle Assisted Chromatin Enrichment) to isolate global and regional chromatin components with high specificity and sensitivity, and SPACEmap to identify the chromatin-contact regions in proteins. Applied to mouse embryonic stem cells, SPACE identified 1,459 chromatin-associated proteins, [~]48% of which are annotated as RBPs, indicating their dual roles in chromatin and RNA-binding. Additionally, SPACEmap stringently verified chromatin-binding of 404 RBPs and identified their chromatin-contact regions. Notably, SPACEmap showed that about half of the caRBPs bind to chromatin by intrinsically disordered regions (IDRs). Studying SPACE and total proteome dynamics from mES cells grown in 2iL and serum medium indicates significant correlation (R = 0.62). One of the most dynamic caRBPs is Dazl, which we find co-localized with PRC2 at transcription start sites of genes that are distinct from Dazl mRNA binding. Dazl and other PRC2-colocalised caRBPs are rich in intrinsically disordered regions (IDRs), which could contribute to the formation and regulation of phase-separated PRC condensates. Together, our approach provides an unprecedented insight into IDR-mediated interactions and caRBPs with moonlighting functions in native chromatin. Rapid expansion of SARS-CoV-2 variants of concern is a result of adaptive epistasis The SARS-CoV-2 pandemic recently entered an alarming new phase with the emergence of the variants of concern (VOC) and understanding their biology is paramount to predicting future ones. Current efforts mainly focus on mutations in the spike glycoprotein (S), but changes in other regions of the viral proteome are likely key. We analyzed more than 900,000 SARS-CoV-2 genomes with a computational systems biology approach including a haplotype network and protein structural analyses to reveal lineage-defining mutations and their critical functional attributes. Our results indicate that increased transmission is promoted by epistasis, i.e., combinations of mutations in S and other viral proteins. Mutations in the non-S proteins involve immune-antagonism and replication performance, suggesting convergent evolution. Furthermore, adaptive mutations appear in geographically disparate locations, suggesting that either independent, repeat mutation events or recombination among different strains are generating VOC. We demonstrate that recombination is a stronger hypothesis, and may be accelerating the emergence of VOC by bringing together cooperative mutations. This emphasizes the importance of a global response to stop the COVID-19 pandemic. Three membrane fusion pore families determine the pathway to pore dilation During exocytosis secretory vesicles fuse with a target membrane and release neurotransmitters, hormones or other bioactive molecules through a membrane fusion pore. The initially small pore may subsequently dilate for full contents release, as commonly observed in amperometric traces. The size, shape and evolution of the pore is critical to the course of contents release, but exact fusion pore solutions accounting for membrane tension and bending energy constraints have not been available. Here we obtained exact solutions for fusion pores between two membranes. We find three families: a narrow pore, a wide pore and an intermediate tether-like pore. For high tensions these are close to the catenoidal and tether solutions recently reported for freely hinged membrane boundaries. We suggest membrane fusion initially generates a stable narrow pore, and the dilation pathway is a transition to the stable wide pore family. The unstable intermediate pore is the transition state that sets the energy barrier for this dilation pathway. Pore dilation is mechanosensitive, as the energy barrier is lowered by increased membrane tension. Finally, we study fusion pores in nanodiscs, powerful systems for the study of individual pores. We show that nanodiscs stabilize fusion pores by locking them into the narrow pore family.

SignificanceDuring neurotransmission, hormone release and other fundamental processes, secretory vesicles fuse their membranes with target membranes to release contents through an initially small membrane fusion pore that subsequently dilates. Dilation is assisted by proteins such as SNAREs and synaptotagmin. While macroscopic soap film shapes are well characterized, finding exact solutions for microscopic cellular membrane surfaces is made more complex by bending energy constraints. Here, computational analysis revealed three families of fusion pores between two membranes. Our work suggests membrane fusion generates a member of the narrow pore family, and pore dilation is a transition to the wide pore family. The energy barrier that SNAREs or synaptotagmin must surmount to achieve dilation is set by a third unstable intermediate pore family.