title: Phage infection restores PQS signaling and enhances growth of a Pseudomonas aeruginosa lasI quorum-sensing mutant

abstract:
Bacteriophage (phage) therapy is reemerging as a valuable tool to combat multidrug resistant bacteria. A major hurdle in developing efficacious bacteriophage therapies is that bacteria acquire resistance to phage killing. In this context, it is noteworthy that quorum sensing (QS), the bacterial cell-to-cell communication mechanism that promotes collective undertaking of group behaviors including anti-phage defenses, enhances bacterial survival in the face of phage attack. QS relies on the production, release, accumulation, and detection of signal molecules called autoinducers. In the opportunistic pathogen Pseudomonas aeruginosa, the LasI/R QS system induces the RhlI/R QS system, and these two systems control, in opposing manners, the PQS QS system that relies on the autoinducer called PQS. A {Delta}lasI mutant is impaired in PQS synthesis, leading to accumulation of the precursor molecule HHQ. HHQ suppresses growth of the P. aeruginosa {Delta}lasI strain. We uncover a phage infection-induced mechanism that restores expression of the pqsH gene in the P. aeruginosa {Delta}lasI QS mutant. PqsH converts HHQ into PQS, preventing HHQ-mediated growth inhibition. Thus, phage-infected P. aeruginosa {Delta}lasI cells exhibit superior growth compared to uninfected cells. Phage infection also restores expression of virulence factors and the CRISPR-cas anti-phage defense system in the P. aeruginosa {Delta}lasI strain. This study highlights a challenge for phage therapy, namely that phage infection may make particular bacterial strains faster growing, more virulent, and resistant to phage killing.

ImportanceThe emergence of multidrug resistant bacteria necessitates development of new antimicrobial therapies. Phage therapy relies on exploiting phages, natural enemies of bacteria, in the fight against pathogenic bacteria. For successful phage therapy development, potent phages that exhibit low propensity for acquisition of bacterial resistance are desired. Here, we show that phage infection restores QS, a cell-to-cell communication mechanism in a P. aeruginosa QS mutant, which increases its virulence and resistance to phage killing. Importantly, clinical isolates of P. aeruginosa frequently harbor mutations in particular QS genes. Thus, phage therapies against such P. aeruginosa strains may inadvertently increase bacterial virulence. Our study underscores the importance of characterizing phage-host interactions in the context of bacterial mutants that are relevant in clinical settings prior to selecting phages for therapy.


title: Mechanisms and fluid dynamics of foraging in heterotrophic nanoflagellates

abstract: Heterotrophic nanoflagellates are the main consumers of bacteria and picophytoplankton in the ocean. In their micro-scale world, viscosity impedes predator-prey contact, and the mechanisms that allow flagellates to daily clear a volume of water for prey corresponding to 106 times their own volume is unclear. It is also unclear what limits observed maximum ingestion rates of about 104 bacterial prey per day. We used high-speed video-microscopy to describe feeding flows, flagellum kinematics, and prey searching, capture, and handling in four species with different foraging strategies. In three species, prey-handling times limit ingestion rates and account well for their reported maximum values. Similarly, observed feeding flows match reported clearance rates. Simple point-force models allowed us to estimate the forces required to generate the feeding flows, between 4-13 pN, and consistent with the force produced by the hairy (hispid) flagellum, as estimated using resistive force theory. Hispid flagella can produce a force that is much higher than the force produced by a naked flagellum with similar kinematics, and the hairy flagellum is therefore key to foraging in most nanoflagellates. Our findings provide a mechanistic underpinning of observed functional responses of prey ingestion rates in nanoflagellates.

category: biophysics


title: Decreased thermal tolerance as a trade-off of antibiotic resistance

abstract: Evolutionary theory predicts that adaptations, including antibiotic resistance, should come with associated fitness costs; yet, many resistance mutations seemingly contradict this prediction by inducing no growth rate deficit. However, most growth assays comparing sensitive and resistant strains have been performed under a narrow range of environmental conditions, which do not reflect the variety of contexts that a pathogenic bacterium might encounter when causing infection. We hypothesized that reduced niche breadth, defined as diminished growth across a diversity of environments, can be a cost of antibiotic resistance. Specifically, we test whether chloramphenicol-resistant Escherichia coli incur disproportionate growth deficits in novel thermal conditions. Here we show that chloramphenicol-resistant bacteria have greater fitness costs at novel temperatures than their antibiotic-sensitive ancestors. In several cases, we observed no resistance cost in growth rate at the historic temperature but saw diminished growth at warmer and colder temperatures. These results were consistent across various genetic mechanisms of resistance. Thus, we propose that decreased thermal niche breadth is an under-documented fitness cost of antibiotic resistance. Furthermore, these results demonstrate that the cost of antibiotic resistance shifts rapidly as the environment changes; these context-dependent resistance costs should select for the rapid gain and loss of resistance as an evolutionary strategy.

category: microbiology


title: Cancer-associated fibroblasts actively compress cancer cells and modulate mechanotransduction

abstract: During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule is a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.

category: cell biology


title: Cytoplasmic pressure maintains epithelial integrity and inhibits cell motility

abstract: Cytoplasmic pressure, a function of actomyosin contractility and water flow, can regulate cellular morphology and dynamics. In mesenchymal cells, cytoplasmic pressure powers cell protrusion through physiological three-dimensional extracellular matrices. However, the role of intracellular pressure in epithelial cells is relatively unclear. Here we find that high cytoplasmic pressure is necessary to maintain barrier function, one of the hallmarks of epithelial homeostasis. Further, our data show that decreased cytoplasmic pressure facilitates lamellipodia formation during the epithelial to mesenchymal transition (EMT). Critically, activation of the actin nucleating protein Arp2/3 is required for the reduction in cytoplasmic pressure and lamellipodia formation in response to treatment with hepatocyte growth factor (HGF) to induce EMT. Thus, elevated cytoplasmic pressure functions to maintain epithelial tissue integrity, while reduced cytoplasmic pressure triggers lamellipodia formation and motility during HGF-dependent EMT.


title: An aging clock using metabolomic CSF

abstract: Quantifying the physiology of aging is essential for improving our understanding of age-related disease and the heterogeneity of healthy aging. Recent studies have shown that in regression models using "-omic" platforms to predict chronological age, residual variation in predicted age is correlated with health outcomes, and suggest that these "omic clocks" provide measures of biological age. This paper presents predictive models for age using metabolomic profiles of cerebrospinal fluid from healthy human subjects, and finds that metabolite and lipid data are generally able to predict chronological age within 10 years. We use these models to predict the age of a cohort of subjects with Alzheimers and Parkinsons disease and find an increase in prediction error, potentially indicating that the relationship between the metabolome and chronological age differs with these diseases. In our analysis of control subjects, we find the carnitine shuttle, sucrose, biopterin, vitamin E metabolism, tryptophan, and tyrosine to be the most associated with age. We showcase the potential usefulness of age prediction models in a small dataset (n = 85), and discuss techniques for drift correction, missing data imputation, and regularized regression which can be used to help mitigate the statistical challenges that commonly arise in this setting. To our knowledge, this work presents the first multivariate predictive metabolomic and lipidomic models for age using mass spectrometry analysis of cerebrospinal fluid.

category: systems biology


title: Non-invasive human skin transcriptome analysis using mRNA in skin surface lipids

abstract: Non-invasive acquisition of mRNA data from the skin would be extremely useful for understanding skin physiology and diseases. Inspired by the holocrine process, in which the sebaceous glands secrete cell contents into the sebum, we focused on the possible presence of mRNAs in skin surface lipids (SSLs). We found that measurable human mRNAs exist in SSLs, where sebum protects them from degradation by RNases. The AmpliSeq transcriptome analysis was modified to measure SSL-RNAs, and our results revealed that SSL-RNAs predominantly contained mRNAs derived from sebaceous glands, epidermis, and hair follicles. Analysis of SSL-RNAs non-invasively collected from patients with atopic dermatitis revealed significantly increased expression of inflammation-related genes and decreased expression of terminal differentiation-related genes, consistent with the results of previous reports. Further, we found that lipid synthesis-related genes were downregulated in the sebaceous glands of patients with atopic dermatitis. These results indicate that the analysis of SSL-RNAs is promising to understand the pathophysiology of skin diseases.

category: molecular biology


title: Behavior of Neural Cells Post Manufacturing and After Prolonged Encapsulation within Conductive Graphene-Laden Alginate Microfibers

abstract: Engineering conductive 3D cell scaffoldings offer unique advantages towards the creation of physiologically relevant platforms with integrated real-time sensing capabilities. Toward this goal, rat dopaminergic neural cells were encapsulated into graphene-laden alginate microfibers using a microfluidic fiber fabrication approach, which is unmatched for creating continuous, highly tunable microfibers. Incorporating graphene increases the conductivity of the alginate microfibers 148%, creating a similar conductivity to native brain tissue. Graphene leads to an increase in the cross-sectional sizes and porosities of the fibers, while reducing the roughness of the fiber surface. The cell encapsulation procedure has an efficiency rate of 50%, and of those cells, approximately 30% remain for the entire 6-day observation period. To understand how encapsulation effects cell genetics, the genes IL-1{beta}, TH, TNF-, and TUBB-3 are analyzed, both after manufacturing and after encapsulation for six days. The manufacturing process and combination with alginate leads to an upregulation of TH, and the introduction of graphene further increases its levels; however, the inverse trend is true of TUBB-3. Long-term encapsulation shows continued upregulation of TH and of TNF-, and six-day exposure to graphene leads to the upregulation of TUBB-3 and IL-1{beta}, which indicates increased inflammation.

category: bioengineering


title: Preparation of "stress-free" concanavalin A-conjugated Dynabeads magnetic beads for CUT&Tag

abstract: Epigenome research has employed various methods to identify genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag(R)Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads(R) magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

category: bioinformatics


title: Design considerations for workflow management systems use in production genomics research and the clinic

abstract:
BackgroundThe changing landscape of genomics research and clinical practice has created a need for computational pipelines capable of efficiently orchestrating complex analysis stages while handling large volumes of data across heterogeneous computational environments. Workflow Management Systems (WfMSs) are the software components employed to fill this gap.

ResultsThis work provides an approach and systematic evaluation of key features of popular bioinformatics WfMSs in use today: Nextflow, CWL, and WDL and some of their executors, along with Swift/T, a workflow manager commonly used in high-scale physics applications. We employed two use cases: a variant-calling genomic pipeline and a scalability-testing framework, where both were run locally, on an HPC cluster, and in the cloud. This allowed for evaluation of those four WfMSs in terms of language expressiveness, modularity, scalability, robustness, reproducibility, interoperability, ease of development, along with adoption and usage in research labs and healthcare settings. This article is trying to answer, "which WfMS should be chosen for a given bioinformatics application regardless of analysis type?".

ConclusionsThe choice of a given WfMS is a function of both its intrinsic language and engine features. Within bioinformatics, where analysts are a mix of dry and wet lab scientists, the choice is also governed by collaborations and adoption within large consortia and technical support provided by the WfMS team/community. As the community and its needs continue to evolve along with computational infrastructure, WfMSs will also evolve, especially those with permissive licenses that allow commercial use. In much the same way as the dataflow paradigm and containerization are now well understood to be very useful in bioinformatics applications, we will continue to see innovations of tools and utilities for other purposes, like big data technologies, interoperability, and provenance.


title: Single-Molecule Tracking of Chromatin-Associated Proteins in the C. elegans Gonad

abstract: 1.Biomolecules are distributed within cells by molecular-scale diffusion and binding events that are invisible in standard fluorescence microscopy. These molecular search kinetics are key to understanding nuclear signaling and chromosome organization, and can be directly observed by single-molecule tracking microscopy. Here, we report a method to track individual proteins within intact C. elegans gonads and apply it to study the molecular dynamics of the axis, a proteinaceous backbone that organizes meiotic chromosomes. Using either fluorescent proteins or enzymatically ligated dyes, we obtain multi-second trajectories with a localization precision of 15-25 nm in nuclei actively undergoing meiosis. Correlation with a reference channel allows for accurate measurement of protein dynamics, compensating for movements of the nuclei and chromatin within the gonad. We find that axis proteins exhibit either static binding to chromatin or free diffusion in the nucleoplasm, and we separately quantify the motion parameters of these distinct populations. Freely diffusing axis proteins selectively explore chromatin-rich regions, suggesting they are circumventing the central phase-separated region of the nucleus. This work demonstrates that single-molecule microscopy can infer nanoscale-resolution dynamics within living tissue, expanding the possible applications of this technique.


title: Development of a mini-replicon-based reverse-genetics system for rice stripe tenuivirus

abstract:
Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in human and livestock, and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing RSV genomic RNA3 eGFP reporter (MR3(-)eGFP), a nucleocapsid (NP), and a codon usage optimized RNA-dependent RNA polymerase (RdRpopt), respectively. Using this mini-replicon system we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of NSs and P19-HcPro-{gamma}b. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis-element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that this mini-replicon system described here will allow the studies of RSV replication, transcription, cell-to-cell movement and host machinery underpinning RSV infection in plants.

IMPORTANCEPlant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into 3 genera: Orthotospovirus, Tenuivirus and Emaravirus. The reverse-genetics systems have been established for members in the genera Orthotospovirus and Emaravirus, respectively. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damages to rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plant.

category: pathology


title: Cell-type specific circadian bioluminescence rhythms recorded from Dbp reporter mice reveal circadian oscillator misalignment

abstract: Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

category: physiology


title: Cell-type specific circadian bioluminescence rhythms in Dbp reporter mice


title: Carbon catabolite repression in pectin digestion by phytopathogen Dickeya dadantii

abstract: The catabolism of pectin from the plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce a massive pectinolytic activity during the maceration phase. While previous studies identified the role of a positive feedback loop specific to the pectin degradation pathway, here we show that the pel expression pattern is controlled by a metabolic switch between glucose and pectin. We develop a dynamical and quantitative regulatory model of this process integrating the two main regulators CRP and KdgR related to these two sources of carbon, and reproducing the concentration profiles of the associated metabolites, cAMP and KDG respectively, quantified using a new HPLC method. The model involves only 5 adjustable parameters, and recapitulates the dynamics of these metabolic pathways during bacterial growth together with the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights their activity as an instance of carbon catabolite repression occurring at the transcriptional regulatory level, and directly related to the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitative different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, explaining their evolutionary conservation as separate genes in this species.


title: Species boundaries among extremely diverse and sexually dimorphic Arrenurus water mites (Acariformes: Hydrachnidiae: Arrenuridae)

abstract: Arrenurus (Arrenuridae) is the most species-rich genus of mites with about 950 named species that inhabit standing, and to a lesser extent, running water habitats around the world. To date, distinguishing species of Arrenurus has been based on male reproductive morphology. Here, we use morphological and molecular approaches to examine species boundaries among 42 named species of Arrenurus, including four named species that have colour variants (red and green A. americanus, and red and blue A. intermedius, A. manubriator and A. apetiolatus), and two unnamed morphospecies. In this study, we examine male genital structures with the use of SEM techniques, and apply mitochondrial (COI barcode region) and nuclear (28S rRNA) gene fragments to test whether male morphology reflects species boundaries in Arrenurus assessed by molecular analyses. Our results reveal that male reproductive morphology parallels species boundaries as judged by molecular data. We discuss the cases of genetically poorly diversified, yet morphologically clearly defined named species. Moreover, we show that based on the species we examined, colour morphs within otherwise morphologically similar specimens represent within-species variation and, in the absence of other diagnostic traits, colour itself can be misleading in distinguishing species. Our outcomes on molecular taxonomy of Arrenurus provide a background for testing hypotheses about speciation rate in water mites.

category: zoology


title: Distinct in vivo dynamics of excitatory synapses onto cortical pyramidal neurons and inhibitory interneurons

abstract: Cortical function relies on the balanced activation of excitatory and inhibitory neurons. However, little is known about the organization and dynamics of shaft excitatory synapses onto cortical inhibitory interneurons, which cannot be easily identified morphologically. Here, we fluorescently visualize the excitatory postsynaptic marker PSD-95 at endogenous levels as a proxy for excitatory synapses onto layer 2/3 pyramidal neurons and parvalbumin-positive (PV+) inhibitory interneurons in the mouse barrel cortex. Longitudinal in vivo imaging reveals that, while synaptic weights in both neuronal types are log-normally distributed, synapses onto PV+ neurons are less heterogeneous and more stable. Markov-model analyses suggest that the synaptic weight distribution is set intrinsically by ongoing cell type-specific dynamics, and substantial changes are due to accumulated gradual changes. Synaptic weight dynamics are multiplicative, i.e., changes scale with weights, though PV+ synapses also exhibit an additive component. These results reveal that cell type-specific processes govern cortical synaptic strengths and dynamics.

category: neuroscience


title: Localizing Brain Function Based on Full Multivariate Activity Patterns: The Case of Visual Perception and Emotion Decoding

abstract: Its now common to approach questions about information representation in the brain using multivariate statistics and machine learning methods. What is less recognized is that, in the process, the ability to perform data-driven discovery and functional localization has diminished. This is because multivariate pattern analysis (MVPA) studies tend to restrict themselves to regions of interest and severely-filtered data, and sound parameter mapping inference is lacking. Here, reproducible evidence is presented that a high-dimensional, brain-wide multivariate linear method can better detect and characterize the occurrence of visual and socio-affective states in a task-oriented functional magnetic resonance imaging (fMRI) experiment; in comparison to the classical localizationist correlation analysis. Classification models for a group of human participants and existing rigorous cluster inference methods are used to construct group anatomical-statistical parametric maps, which correspond to the most likely neural correlates of each psychological state. This led to the discovery of a multidimensional pattern of brain activity which reliably encodes for the perception of happiness in the visual cortex, cerebellum and some limbic areas. We failed to find similar evidence for sadness and anger. Anatomical consistency of discriminating features across subjects and contrasts despite of the high number of dimensions, as well as agreement with the wider literature, suggest MVPA is a viable tool for full-brain functional neuroanatomical mapping and not just prediction of psychological states. The present work paves the way for future functional brain imaging studies to provide a complementary picture of brain functions (such as emotion), according to their macroscale dynamics.


title: Bipartite functional fractionation within the neural system for social cognition supports the psychological continuity of self vs. other

abstract: Research of social neuroscience establishes that regions in the brains default network (DN) and semantic network (SN) are engaged by socio-cognitive tasks. Research of the human connectome shows that DN and SN regions are both situated at the high-order end of cortical gradient but differ in their positions on this gradient. In the present study, we integrated these two bodies of research, used the psychological continuity of self vs. other as a  test-case, and used fMRI to investigate whether these networks would encode social concepts differently. We found a robust dissociation between the DN and SN - while both networks contained sufficient information for decoding broad-stroke distinction of social categories, the DN carried more generalisable information for cross-classifying across social distance and emotive valence than did the SN. We also found that the overarching distinction of self vs. other was a principal divider of the representational space while social distance was an auxiliary factor (subdivision, nested within the principal dimension), and this representational landscape was more manifest in the DN than in the SN. Taken together, our findings demonstrate how insights from connectome research can benefit social neuroscience, and have implications for clarifying the two networks differential contributions to social cognition.


title: Asymmetric learning facilitates human inference of transitive relations

abstract: Humans and other animals are capable of inferring never-experienced relations (e.g., A>C) from other relational observations (e.g., A>B and B>C). The processes behind such transitive inference are subject to intense research. Here, we demonstrate a new aspect of relational learning, building on previous evidence that transitive inference can be accomplished through simple reinforcement learning mechanisms. We show in simulations that inference of novel relations benefits from an asymmetric learning policy, where observers update only their belief about the winner (or loser) in a pair. Across 4 experiments (n=145), we find substantial empirical support for such asymmetries in inferential learning. The learning policy favoured by our simulations and experiments gives rise to a compression of values which is routinely observed in psychophysics and behavioural economics. In other words, a seemingly biased learning strategy that yields well-known cognitive distortions can be beneficial for transitive inferential judgments.


title: Stimulation with mycobacterial glycolipids and PPD reveals different innate immune response profiles in active and latent TB

abstract: Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and PPD. At 24 h and 5 days of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM mainly affected antigen-presenting cells to produce both proinflammatory (IL-2, IL-6, IL-17A, TNF- and GM-CSF), and IL-4 and IL-10 cytokines, but not IFN-{gamma}. LAM triggered a similar, albeit weaker, response. By contrast, PPD induced an increase in IFN-{gamma}-producing cells. Moreover, PPD also led to increased numbers of IL-2, IL-6, IL-10, IL-17A, TNF- and GrzB-producing cells. Treatment with an anti-TLR2 antibody led to partial inhibition of PIM-induced IL-6 production in myeloid cells, suggesting that PIM induces IL-6 production through TLR2. Expansion of monocyte subsets in response to PIM or LAM was reduced in both ATB and LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern in Mtb-infected individuals.

category: immunology


title: Relish plays a dynamic role in the niche to modulate Drosophila bloodprogenitor homeostasis in development and infection.

abstract: Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. Here, we show that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-{kappa}B (NF-{kappa}B), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program towards precocious differentiation, thereby bolstering the cellular arm of the immune response.

category: developmental biology


title: Substitutions and codon usage in SARS-CoV-2 in mammals indicate natural selection and host adaptation

abstract: The outbreak of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, rapidly spread to create a global pandemic and has continued to spread across hosts from humans to animals, transmitting particularly effectively in mink. How SARS-CoV-2 evolves in animals and humans and the differences in the separate evolutionary processes remain unclear. We analyzed the composition and codon usage bias of SARS-CoV-2 in infected humans and animals. Compared with other animals, SARS-CoV-2 in mink had the most substitutions. The substitutions of cytidine in SARS-CoV-2 in mink account for nearly 50% of the substitutions, while those in other animals represent only 30% of the substitutions. The incidence of adenine transversion in SARS-CoV-2 in other animals is threefold higher than that in mink-CoV (the SARS-CoV-2 virus in mink). A synonymous codon usage analysis showed that SARS-CoV-2 is optimized to adapt in the animals in which it is currently reported, and all the animals showed decreased adaptability relative to that of humans, except for mink. A binding affinity analysis indicated that the spike protein of the SARS-CoV-2 variant in mink showed a greater preference for binding with the mink receptor ACE2 than with the human receptor, especially as the mutation Y453F and F486L in mink-CoV lead to improvement of binding affinity for mink receptor. Our study focuses on the divergence of SARS-CoV-2 genome composition and codon usage in humans and animals, indicating possible natural selection and current host adaptation.

category: evolutionary biology


title: The testis-specific transcription factor TCFL5 responds to A MYBto elaborate the male meiotic program in placental mammals

abstract: In male mice, the transcription factor (TF) A-MYB initiates reprogramming of gene expression after spermatogonia enter meiosis. We report that A-MYB activates Tcfl5, a testis-specific TF first produced in pachytene spermatocytes. Subsequently, A-MYB and TCFL5 reciprocally reinforce their own transcription to establish an extensive circuit that regulates meiosis. TCFL5 promotes transcription of genes required for mRNA turnover, pachytene piRNA production, meiotic exit, and spermiogenesis. This transcriptional architecture is conserved in rhesus macaque, suggesting TCFL5 plays a central role in meiosis and spermiogenesis in placental mammals. Tcfl5em1/em1 mutants are sterile, and spermatogenesis arrests at the mid- or late-pachytene stage of meiosis.

category: genetics


title: MetaMutationalSigs: Comparison of mutational signature refitting results made easy

abstract:
SummaryThe analysis of mutational signatures is becoming increasingly common in cancer genetics, with emerging implications in cancer evolution, classification, treatment decision and prognosis. Recently, several packages have been developed for mutational signature analysis, with each using different methodology and yielding significantly different results. Because of the nontrivial differences in tools refitting results, researchers may desire to survey and compare the available tools, in order to objectively evaluate the results for their specific research question, such as which mutational signatures are prevalent in different cancer types. There is a need for a software that can aggregate results from different refitting packages and present them in a user-friendly way to facilitate effective comparison of mutational signatures.

Availability and implementationMetaMutationalSigs is implemented using R and python and is available for installation using Docker and available at: https://github.com/EESI/MetaMutationalSigs

ContactGail Rosen (glr26@drexel.edu).

Supplementary informationMore information about the package including test data and results are available at https://github.com/EESI/MetaMutationalSigs


title: Ensuring scientific reproducibility in bio-macromolecular modeling via extensive, automated benchmarks

abstract: Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met. We describe the implementation of a test server framework and 40 scientific benchmarks, covering numerous applications in Rosetta bio-macromolecular modeling. High performance computing cluster integration allows these benchmarks to run continuously and automatically. Detailed protocol captures are useful for developers and users of Rosetta and other macromolecular modeling tools. The framework and design concepts presented here are valuable for developers and users of any type of scientific software and for the scientific community to create reproducible methods. Specific examples highlight the utility of this framework and the comprehensive documentation illustrates the ease of adding new tests in a matter of hours.


title: Expression Based Species Deconvolution and Realignment Removes Misalignent Error in Multi-species Single Cell Data

abstract: Although single-cell RNA sequencing of xenograft samples has been widely used, no comprehensive bioinformatics pipeline is available for human and mouse mixed single-cell analyses. Considering the numerous homologous genes across the human and mouse genomes, misalignment errors should be evaluated, and a new algorithm is required. We assessed the extents and effects of misalignment errors when using human and mouse combined reference data and developed a new bioinformatics pipeline with expression-based species deconvolution to minimize errors. We also evaluated false-positive signals for a species presumed to originate from ambient RNA of the other species and applied a computational method to remove them. Misaligned reads account for an average of 0.5% of total reads, but such reads were concentrated to few genes that were greatly affected. Human and mouse mixed single-cell data, analyzed using our pipeline, clustered well with unmixed data and showed higher k-nearest-neighbor batch effect test and Local Inverse Simpsons Index scores than those derived from Cell Ranger. We also applied our pipeline to multispecies multisample single-cell library containing breast cancer xenograft tissue and successfully identified all samples using genomic array and expression. Moreover, diverse cell types in the tumor microenvironment were well captured. We present our bioinformatics pipeline for mixed human and mouse single-cell data, which can also be applied to pooled libraries to obtain cost-effective single-cell data. We also address major consideration points when analyzing multispecies single-cell data for other applications.


title: Magnetic wire active microrhreology of human respiratory mucus

abstract: Mucus is a viscoelastic gel secreted by the pulmonary epithelium in the tracheobronchial region of the lungs. The coordinated beating of cilia in contact with the gel layer moves mucus upwards towards pharynx, removing inhaled pathogens and particles from the airways. The efficacy of this clearance mechanism depends primarily on the rheological properties of mucus. Here we use a magnetic wire based microrheology technique to study the viscoelastic properties of human mucus collected from human bronchus tubes. The response of wires between 5 and 80 {micro}m in length to a magnetic rotating field is monitored by optical time-lapse microscopy and analyzed using constitutive equation models of rheology, including Maxwell and Kelvin-Voigt. The static shear viscosity and elastic modulus can be inferred from low frequency (10-3 - 10 rad s-1) measurements, leading to the evaluation of the mucin network relaxation time. This relaxation time is found to be widely distributed, from one to several hundred seconds. Mucus is identified as a viscoelastic liquid with an elastic modulus of 2.5 {+/-} 0.5 Pa and a static viscosity of 100 {+/-} 40 Pa s. Our work shows that beyond the established spatial variations in rheological properties due to microcavities, mucus exhibits secondary inhomogeneities associated with the relaxation time of the mucin network that may be important for its flow properties.


title: Magnetic wire active microrheology of human respiratory mucus


title: COVID-19 mRNA vaccines drive differential Fc-functional profiles in pregnant, lactating, and non-pregnant women

abstract: Significant immunological changes occur throughout pregnancy to tolerize the mother and allow growth of the fetal graft. However, additional local and systemic immunological adaptations also occur, allowing the maternal immune system to continue to protect the dyad against foreign invaders both during pregnancy and after birth through lactation. This fine balance of tolerance and immunity, along with physiological and hormonal changes, contribute to increased susceptibility to particular infections in pregnancy, including more severe COVID-19 disease. Whether these changes also make pregnant women less responsive to vaccination or induce altered immune responses to vaccination remains incompletely understood. To holistically define potential changes in vaccine response during pregnancy and lactation, we deeply profiled the humoral vaccine response in a group of pregnant and lactating women and non-pregnant age-matched controls. Vaccine-specific titers were comparable, albeit slightly lower, between pregnant and lactating women, compared to non-pregnant controls. Among pregnant women, we found higher antibody titers and functions in those vaccinated with the Moderna vaccine. FcR-binding and antibody effector functions were induced with delayed kinetics in both pregnant and lactating women compared to non-pregnant women. Antibody boosting resulted in high FcR-binding titers in breastmilk. These data point to an immune resistance to generate highly inflammatory antibodies during pregnancy and lactation, and a critical need to follow prime/boost timelines in this vulnerable population to ensure full immunity is attained.


title: Twelve-month specific IgG response to SARS-CoV-2 receptor-binding domain among COVID-19 convalescent plasma donors in Wuhan

abstract: To investigate the duration of humoral immune response in convalescent coronavirus disease 2019 (COVID-19) patients, we conduct a 12-month longitudinal study through collecting a total of 1,782 plasma samples from 869 convalescent plasma donors in Wuhan, China and test specific antibody responses. The results show that positive rate of IgG antibody against receptor-binding domain of spike protein (RBD-IgG) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the COVID-19 convalescent plasma donors exceeded 70% for 12 months post diagnosis. The level of RBD-IgG decreases with time, with the titer stabilizing at 64.3% of the initial level by the 9th month. Moreover, male plasma donors produce more RBD-IgG than female, and age of the patients positively correlates with the RBD-IgG titer. A strong positive correlation between RBD-IgG and neutralizing antibody titers is also identified. These results facilitate our understanding of SARS-CoV-2-induced immune memory to promote vaccine and therapy development.


title: Driving potent neutralization of a SARS-CoV-2 Variant of Concern with a heterotypic boost

abstract: The emergence of SARS-CoV-2 Variants of Concern (VOCs) with mutations in key neutralizing antibody epitopes threatens to undermine vaccines developed against the pandemic founder variant (Wu-Hu-1). Widespread vaccine rollout and continued transmission are creating a population that has antibody responses of varying potency to Wu-Hu-1. Against this background, it is critical to assess the outcomes of subsequent immunization with variant antigens. It is not yet known whether heterotypic vaccine boosts would be compromised by original antigenic sin, where pre-existing responses to a prior variant dampen responses to a new one, or whether the primed memory B cell repertoire would bridge the gap between Wu-Hu-1 and VOCs. Here, we show that a single adjuvanted dose of receptor binding domain (RBD) protein from VOC 501Y.V2 (B.1.351) drives an extremely potent neutralizing antibody response capable of cross-neutralizing both Wu-Hu-1 and 501Y.V2 in rhesus macaques previously immunized with Wu-Hu-1 spike protein. Passive immunization with plasma sampled following this boost protected K18-hACE2 mice from lethal challenge with a 501Y.V2 clinical isolate, whereas only partial protection was afforded by plasma sampled after two Wu-Hu-1 spike immunizations.


title: XAV-19, a novel swine glyco-humanized polyclonal antibody against SARS-CoV-2 spike, efficiently neutralizes B.1.1.7 British and B.1.351 South-African variants.

abstract: Amino acid substitutions and deletions in Spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can reduce the effectiveness of monoclonal antibodies (mAbs). In contrast, heterologous polyclonal antibodies raised against S protein, through the recognition of multiple target epitopes, have the potential to maintain neutralization capacities. XAV-19 is a swine glyco-humanized polyclonal neutralizing antibody raised against the receptor binding domain (RBD) of the Wuhan-Hu-1 Spike protein of SARS-CoV-2. XAV-19 target epitopes were found distributed all over the RBD and particularly cover the receptor binding motives (RBM), in direct contact sites with the Angiotensin Converting Enzyme-2 (ACE-2). Therefore, in Spike/ACE2 interaction assays, XAV-19 showed potent neutralization capacities of the original Wuhan Spike and of the United Kingdom (Alpha/B.1.1.7) and South African (Beta/B.1.351) variants. These results were confirmed by cytopathogenic assays using Vero E6 and live virus variants including the Brazil (Gamma/P.1) and the Indian (Delta/B.1.617.2) variants. In a selective pressure study with the Beta strain on Vero E6 cells conducted over 1 month, no mutation was associated with addition of increasing doses XAV-19. The potential to reduce viral load in lungs was confirmed in a human ACE2 transduced mouse model. XAV-19 is currently evaluated in patients hospitalized for COVID-19-induced moderate pneumonia in a phase 2a-2b (NCT04453384) where safety was already demonstrated and in an ongoing 2/3 trial (NCT04928430) to evaluate the efficacy and safety of XAV-19 in patients with moderate-to-severe COVID-19. Owing to its polyclonal nature and its glyco-humanization, XAV-19 may provide a novel safe and effective therapeutic tool to mitigate the severity of coronavirus disease 2019 (Covid-19) including the different variants of concern identified so far.


title: XAV-19, a swine glyco-humanized polyclonal antibody against SARS-CoV-2 Spike receptor-binding domain, targets multiple epitopes and broadly neutralizes variants.


title: Co-infection of chickens with H9N2 and H7N9 avian influenza viruses leads to emergence of reassortant H9N9 virus with increased fitness for poultry and enhanced zoonotic potential

abstract:
An H7N9 low pathogenicity avian influenza virus (LPAIV) emerged through genetic reassortment between H9N2 and other LPAIVs circulating in birds in China. This virus causes inapparent clinical disease in chickens, but zoonotic transmission results in severe and fatal disease in humans. We evaluated the consequences of reassortment between the H7N9 and the contemporary H9N2 viruses of G1 lineage that are enzootic in poultry across the Indian sub-continent and the Middle East. Co-infection of chickens with these viruses resulted in emergence of novel reassortant H9N9 viruses carrying genes derived from both H9N2 and H7N9 viruses. These reassortant H9N9 viruses showed significantly increased replication fitness, enhanced pathogenicity in chicken embryos and the potential to transmit via contact among ferrets. Our study highlights that the co-circulation of H7N9 and H9N2 viruses could represent a threat for the generation of novel reassortant viruses with greater virulence in poultry and an increased zoonotic potential.

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C_FIG In BriefH9N2 viruses have a high propensity to reassort with other avian influenza viruses. We found that co-infection of chickens with H9N2 and H7N9 led to the emergence of reassortant viruses including the H9N9 subtype. Some reassortant H9N9 viruses exhibited increased replication fitness, increased pathogenicity in the chicken embryo, greater avidity for human and avian cell receptors, lower pH fusion and contact-transmission to ferrets. This study demonstrated the ability of viruses that already exist in nature to exchange genetic material, highlighting the potential emergence of viruses from these subtypes with increased zoonotic potential. There are nine H9 influenza A subtypes carrying different neuraminidase (NA) genes, including H9N9 viruses, while they are not common they do exist in nature as wildtypes (CDC).

HighlightsO_LICo-infection of chickens with H7N9 and H9N2 led to emergence of reassortant H9N9 viruses
C_LIO_LIReassortant H9N9 viruses had an increased replication rate in avian and human cells
C_LIO_LIReassortant H9N9 viruses had a lower pH fusion and significantly higher receptor binding to  2,3 sialoglycans
C_LIO_LIReassortant H9N9 replicated in ferrets at similar levels compared to H7N9 and transmitted via direct contact
C_LIO_LIFerrets exposed to reassortant H9N9 by aerosol contact were also found to be seropositive
C_LIO_LIExperimental simulation of events that may occur naturally with circulating viruses has demonstrated the risk of emergence of viruses with increased zoonotic potential.
C_LI


title: Identification of diverse targets of arginine phosphorylation in Mycolicibacterium smegmatis by shotgun proteomics

abstract:
Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate of Mtb. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on a diverse collection of targets within the Msm proteome. Physicochemical and functional characterization of targets suggest that arginine phosphorylation is applied in a sequence-independent manner as part of a proteome-wide quality control pathway. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species.

GRAPHICAL ABSTRACT

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title: An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity

abstract:
During the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic.

Graphical Abstract

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title: The Lithuanian reference genome LT1 - a human de novo genome assembly with short and long read sequence and Hi-C data

abstract: We present LT1, the first high-quality human reference genome from the Baltic States. LT1 is a female de novo human reference genome assembly constructed using 57x of ultra-long nanopore reads and 47x of short paired-end reads. We also utilized 72 Gb of Hi-C chromosomal mapping data to maximize the assemblys contiguity and accuracy. LT1s contig assembly was 2.73 Gbp in length comprising of 4,490 contigs with an N50 value of 13.4 Mbp. After scaffolding with Hi-C data and extensive manual curation, we produced a chromosome-scale assembly with an N50 value of 138 Mbp and 4,699 scaffolds. Our gene prediction quality assessment using BUSCO identify 89.3% of the single-copy orthologous genes included in the benchmarking set. Detailed characterization of LT1 suggested it has 73,744 predicted transcripts, 4.2 million autosomal SNPs, 974,000 short indels, and 12,330 large structural variants. These data are shared as a public resource without any restrictions and can be used as a benchmark for further in-depth genomic analyses of the Baltic populations.

category: genomics


title: Dynamic prospect theory - two core economic decision theories coexist in the gambling behavior of monkeys

abstract:
Research in behavioral economics and reinforcement learning has given rise to two influential theories describing human economic choice under uncertainty. The first, prospect theory, assumes that decision-makers use static mathematical functions, utility and probability weighting, to calculate the values of alternatives. The second, reinforcement learning theory, posits that dynamic mathematical functions update the values of alternatives based on experience through reward prediction error (RPE). To date, these theories have been examined in isolation without reference to one another. Therefore, it remains unclear whether RPE affects a decision-makers utility and/or probability weighting functions, or whether these functions are indeed static as in prospect theory. Here, we propose a dynamic prospect theory model that combines prospect theory and RPE, and test this combined model using choice data on gambling behavior of captive macaques. We found that under standard prospect theory, monkeys, like humans, had a concave utility function. Unlike humans, monkeys exhibited a concave, rather than inverse-S shaped, probability weighting function. Our dynamic prospect theory model revealed that probability distortions, not the utility of rewards, solely and systematically varied with RPE: after a positive RPE, the estimated probability weighting functions became more concave, suggesting more optimistic belief about receiving rewards and over-weighted subjective probabilities at all probability levels. Thus, the probability perceptions in laboratory monkeys are not static even after extensive training, and are governed by a dynamic function well captured by the algorithmic feature of reinforcement learning. This novel evidence supports combining these two major theories to capture choice behavior under uncertainty.

Significance statementWe propose and test a new decision theory under uncertainty by combining pre-existing two influential theories in the neuroeconomics: prospect theory from economics and prediction error theory from reinforcement learning. Collecting a large dataset (over 60,000 gambling decisions) from laboratory monkeys enables us to test the hybrid model of these two core decision theories reliably. Our results showed over-weighted subjective probabilities at all probability levels after lucky win, indicating that positive prediction error systematically bias decision-makers more optimistically about receiving rewards. This trial-by-trial prediction-error dynamics in probability perception provides outperformed performance of the model compared to the standard static prospect theory. Thus, both static and dynamic elements coexist in monkeys risky decision-making, an evidence contradicting the assumption of prospect theory.

category: animal behavior and cognition


title: Rational Selection of PCR Primer/Probe Design Sites for SARS-CoV-2

abstract: Various reports of decreased analytical sensitivities of real-time PCR-based detection of Coronavirus Disease 2019 (COVID-19) have been associated with occurrence of mutations in the target area of primer/probe binding. Knowledge about propensities of different genes to undergo mutation can inform researchers to select optimal genes to target for the qPCR design. We analyzed supplementary data from over 45 thousand SARS-CoV-2 genomes provided by Mercatelli et al to calculate the unique and prevalent mutations in different genes of SARS-CoV-2. We found that non-structural proteins in the ORF1ab region were more conserved compared to structural genes. Further factors which need to be relied upon for proper selection of genes for qPCR design are discussed.


title: Nutrient Responsive O-GlcNAcylation Dynamically Modulates Galectin 3 Secretion

abstract: Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and in fact, characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans that are secreted by a poorly characterized non-classical secretory mechanism. Once outside the cell, galectins bind to terminal galactose residues of cell surface glycans and modulate numerous extracellular functions like clathrin independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal. This study shows that galectin 3 is O-GlcNAcylated, and that changes in O-GlcNAc cycling alters its secretion. Moreover, we determined that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions and that these changes were dependent on dynamic O-GlcNAcylation. Finally, we showed that alterations in galectin 3 secretion via disrupted O-GlcNAcylation drove changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function of transducing nutrient sensing information coded in cell surface glycosylation into biological effects.


title: Sobp modulates Six1 transcriptional activation and is required during craniofacial development

abstract:
Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, craniofacial and/or renal defects. Mutations in the transcription factor Six1 and its cofactor Eya1, both required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 cofactor and SOBP mutations in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-IP and immunofluorescence experiments demonstrate that Sobp binds to and co-localizes with Six1 in the cell nucleus. Luciferase assays show that Sobp represses Six1+Eya1 transcriptional activation. Experiments in Xenopus embryos that either knockdown or increase expression show that Sobp is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human mutation disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function, is required for vertebrate craniofacial development, and identifies Sobp as a potential candidate gene for BOR and other deafness syndromes.

Summary statementSobp interacts with Six1 in the cell nucleus and represses the Six1+Eya1 transcriptional activation. In Xenopus embryos, Sobp functions during early stages of inner ear development.


title: The Okur-Chung Neurodevelopmental Syndrome (OCNDS) mutation CK2K198R leads to a rewiring of kinase specificity

abstract: Okur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the CSNK2A1 gene, which encodes the alpha subunit of casein kinase II (CK2). The most frequently occurring mutation is lysine 198 to arginine (K198R). To investigate the impact of this mutation, we first generated a high-resolution phosphorylation motif of CK2WT, including the first characterization of specificity for tyrosine phosphorylation activity. A second high resolution motif representing CK2K198R substrate specificity was also generated. Here we report for the first time the impact of the OCNDS associated CK2K198R mutation. Contrary to prior speculation, the mutation does not result in a loss of function, but rather shifts the substrate specificity of the kinase. Broadly speaking the mutation leads to 1) a decreased preference for acidic residues in the +1 position, 2) a decreased preference for threonine phosphorylation, 3) an increased preference for tyrosine phosphorylation, and 4) an alteration of the tyrosine phosphorylation specificity motif. To further investigate the result of this mutation we have developed a probability-based scoring method, allowing us to predict shifts in phosphorylation in the K198R mutant relative to the wild type kinase. As an initial step we have applied the methodology to the set of axonally localized ion channels in an effort to uncover potential alterations of the phosphoproteome associated with the OCNDS disease condition.


title: Global-scale CRISPR gene editor specificity profiling by ONE-seq identifies population-specific, variant off-target effects

abstract: Defining off-target profiles of gene-editing nucleases and CRISPR base editors remains an important challenge for use of these technologies, therapeutic or otherwise. Existing methods can identify off-target sites induced by these gene editors on an individual genome but are not designed to account for the broad diversity of genomic sequence variation that exists within populations of humans or other organisms. Here we describe OligoNucleotide Enrichment and sequencing (ONE-seq), a novel in vitro method that leverages customizable, high-throughput DNA synthesis technology instead of purified genomic DNA (gDNA) from individual genomes to profile gene editor off-target sites. We show that ONE-seq matches or exceeds the sensitivity of existing single-genome methods for identifying bona fide CRISPR-Cas9 off-target sites in cultured human cells and in vivo in a liver-humanized mouse model. In addition, ONE-seq outperforms existing best-in-class single-genome methods for defining off-target sites of CRISPR-Cas12a nucleases, cytosine base editors (CBEs), and adenine base editors (ABEs), unveiling previously undescribed bona fide off-target sites for all these editors in human cells. Most importantly, we leveraged ONE-seq to generate the first experimentally-derived population-scale off-target profiles for Cas9 nucleases that define the impacts of sequence variants from >2,500 individual human genome sequences in the 1000 Genomes Project database. Notably, some of the variants we identified that lead to increased mutation frequencies at off-target sites are enriched in specific human populations. We validated that novel population-specific, variant-sensitive off-target sites nominated by ONE-seq in vitro can show increased frequencies of mutations in human lymphoblastoid cells (LCLs) harboring these sequence variants. Collectively, our results demonstrate that ONE-seq is a highly sensitive off-target nomination method that can uniquely be used to identify population subgroup-linked differences in off-target profiles of gene editors. ONE-seq provides an important new pathway by which to assess the impacts of global human genetic sequence diversity on the specificities of gene editors, thereby enabling a broader and more all-inclusive approach for profiling off-target effects of these transformative therapeutic technologies.


title: Biochemical alterations precede neurobehavioral deficits in a novel mouse model of Friedreich ataxia

abstract: Friedreich Ataxia (FA) is a rare neuro-cardiodegenerative disease, caused by partial deficiency of frataxin, a mitochondrial protein. This deficiency is caused by the presence of a GAA triplet expansion in the first intron of the frataxin gene or, in some patients, by point mutations. Generating mouse models mimicking FA has been challenging, as this disease is manifested when frataxin levels are below a pathological threshold. In the present work, we have characterized a new mouse model of FA (FXNI151F) based on a pathological point mutation (I154F) present in some FA patients. These mice present very low frataxin levels in all tissues and display neurological deficits resembling those observed in FA patients. We have also observed decreased content of components from OXPHOS complexes I and II, decreased aconitase activity, and alterations in the antioxidant defenses. Remarkably, these biochemical alterations precede the appearance of neurological symptoms and present a different profile in heart and brain or cerebellum. The FXNI151F mouse is an excellent tool for analyzing the consequences of frataxin deficiency in different tissues and for testing new therapies.

category: biochemistry


title: Cerebello-Basal Ganglia Network Global Efficiency Predicts Cortical Network Global Efficiency

abstract: The cerebellum (CB) and basal ganglia (BG) each have topographically distinct functional subregions that are functionally and anatomically interconnected with cortical regions through discrete thalamic loops and with each other via disynaptic connections, with previous work detailing high levels of functional connectivity between these phylogenetically ancient regions. It was posited that this CB-BG network provides support for cortical systems processing, spanning cognitive, emotional, and motor domains, implying subcortical network measures are strongly related to cortical network measures (Bostan & Strick, 2018); however, it is currently unknown how network measures within distinct CB-BG networks relate to cortical network measures. Here, 122 regions of interest comprising cognitive and motor CB-BG networks and 7 canonical cortical resting-state were used to investigate whether the integration (quantified using global efficiency, GE) of cognitive CB-BG network (CCBN) nodes and their segregation from motor CB-BG network (MCBN) nodes is related to cortical network GE and segregation in 233 non-related, right- handed participants (Human Connectome Project-1200). CCBN GE positively correlated with GE in the default mode, motor, and auditory networks and MCBN GE positively correlated with GE in all networks except the default mode and emotional. MCBN segregation was related to MN segregation. These findings highlight the CB-BG networks role in executive function, task switching, and verbal working memory. This work has implications for understanding cortical network organization and cortical-subcortical interactions in healthy adults and may help in deciphering subcortical differences seen in disease states.


title: Cerebello-Basal Ganglia Networks and Cortical Network Global Efficiency


title: Joint representation of working memory and uncertainty in human cortex

abstract: Neural representations of visual working memory (VWM) are noisy, and thus, decisions based on VWM are inevitably subject to uncertainty. However, the mechanisms by which the brain simultaneously represents the content and uncertainty of memory remain largely unknown. Here, inspired by the theory of probabilistic population codes, we test the hypothesis that the human brain represents an item maintained in VWM as a probability distribution over stimulus feature space, thereby capturing both its content and uncertainty. We used a neural generative model to decode probability distributions over memorized locations from fMRI activation patterns. We found that the mean of the probability distribution decoded from retinotopic cortical areas predicted memory reports on a trial-by-trial basis. Moreover, in several of the same mid-dorsal stream areas the spread of the distribution predicted subjective trial-by-trial uncertainty judgments. These results provide evidence that VWM content and uncertainty are jointly represented by probabilistic neural codes.


title: Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance

abstract:
BackgroundNon-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is complex and can be affected by numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognised genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging.

ResultsHere we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain. Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, these regions are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism.

ConclusionOur compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of the human brain and will be relevant to uncover missing heritability in human genetic brain disorders.


title: On the many advantages of using the VariantExperiment class to store, exchange and analyze SARS-CoV-2 genomic data and associated metadata

abstract: On Friday, 19 March 2021, WHO organized a virtual global workshop highlighting the need for a globally coordinated plan to increase SARS-CoV-2 genetic sequencing capacities to detect SARS-CoV-2 mutations and variants, and to monitor virus genomic evolution worldwide. One week later, in another virtual meeting, it focused on sero epidemiology for SARS-CoV-2 variants of concern and variants of interest. Efficient monitoring of the virus relies on the storage, handling and sharing of the genomic data and the associated metadata. In this manuscript, we demonstrate how the Bioconductor VariantExperiment class addresses these needs, offering a robust and efficient solution to the requirements laid out by the WHO.


title: A new SARS-CoV-2 lineage that shares mutations with known Variants of Concern is rejected by automated sequence repository quality control

abstract: We report a SARS-CoV-2 lineage that shares N501Y, P681H, and other mutations with known variants of concern, such as B.1.1.7. This lineage, which we refer to as B.1.x (COG-UK sometimes references similar samples as B.1.324.1), is present in at least 20 states across the USA and in at least six countries. However, a large deletion causes the sequence to be automatically rejected from repositories, suggesting that the frequency of this new lineage is underestimated using public data. Recent dynamics based on 339 samples obtained in Santa Cruz County, CA, USA suggest that B.1.x may be increasing in frequency at a rate similar to that of B.1.1.7 in Southern California. At present the functional differences between this variant B.1.x and other circulating SARS-CoV-2 variants are unknown, and further studies on secondary attack rates, viral loads, immune evasion and/or disease severity are needed to determine if it poses a public health concern. Nonetheless, given what is known from well-studied circulating variants of concern, it seems unlikely that the lineage could pose larger concerns for human health than many already globally distributed lineages. Our work highlights a need for rapid turnaround time from sequence generation to submission and improved sequence quality control that removes submission bias. We identify promising paths toward this goal.


title: Differential effects of cerebellar degeneration on feedforward versus feedback control across speech and reaching movements

abstract:
Errors that result from a mismatch between predicted movement outcomes and sensory afference are used to correct ongoing movements through feedback control and to adapt feedforward control of future movements. The cerebellum has been identified as a critical part of the neural circuit underlying implicit adaptation across a wide variety of movements (reaching, gait, eye movements, and speech). The contribution of this structure to feedback control is less well understood: although it has recently been shown in the speech domain that individuals with cerebellar degeneration produce even larger online corrections for sensory perturbations than control participants, similar behavior has not been observed in other motor domains. Currently, comparisons across domains are limited by different population samples and potential ceiling effects in existing tasks. To assess the relationship between changes in feedforward and feedback control associated with cerebellar degeneration across motor domains, we evaluated adaptive (feedforward) and compensatory (feedback) responses to sensory perturbations in reaching and speech production in human participants of both sexes with cerebellar degeneration and neurobiologically healthy controls. As expected, the cerebellar group demonstrated impaired adaptation in both reaching and speech. In contrast, the groups did not differ in their compensatory response in either domain. Moreover, compensatory and adaptive responses in the cerebellar group were not correlated within or across motor domains. These results point to a general impairment in feedforward control with spared feedback control in cerebellar degeneration. However, the magnitude of feedforward impairments and potential changes in feedback-based control manifest in a domain-specific manner across individuals.

Significance StatementThe cerebellum contributes to feedforward updating of movement in response to sensory errors, but its role in feedback control is less understood. Here, we tested individuals with cerebellar degeneration (CD), using sensory perturbations to assess adaptation of feedforward control and feedback gains during reaching and speech production tasks. The results confirmed that CD leads to reduced adaption in both domains. However, feedback gains were unaffected by CD in either domain. Interestingly, measures of feedforward and feedback control were not correlated across individuals within or across motor domains. Together, these results indicate a general impairment in feedforward control with spared feedback control in CD. However, the magnitude of feedforward impairments manifests in a domain-specific manner across individuals.


title: The viral vectored vaccine targeting Thymosin β10 controls tumour growth in a murine model of prostate cancer

abstract: Direct analysis of MHC-presented antigens by mass spectrometry has been successfully applied for the evaluation of the antigenic landscape of different human tissues and malignancies in the past. In the current study, for the first time this approach has been applied to investigate a murine prostate cancer peptidome for the development of next generation prostate cancer vaccines using the TRansgenic Adenocarcinoma of Mouse Prostate, TRAMP, model. We have performed immunopeptidomic analysis of the normal mouse prostate and mouse prostate adenocarcinoma samples and identified a number of peptides overexpressed on tumour tissue compared to the normal prostate. We selected two of these proteins previously reported to play a role in carcinogenesis, thymosin beta 4 (Thy{beta}4) and thymosin beta 10 (Thy{beta}10), expressed them in the chimpanzee adenovirus (ChAdOx1) and Modified Vaccinia Ankara (MVA) recombinant viral vectors and evaluated these constructs as a candidate prostate cancer vaccine in a murine prostate cancer model. We have found that the vaccine encoding the Thy{beta}10 polypeptide tethered to the MHC class II associated invariant chain, CD74, significantly delayed outgrowth of established tumours in an ectopic TRAMP-C1 mouse model of prostate cancer. The observed tumour-protective efficacy was not associated with Thy{beta}10-specific cellular or antibody responses, suggesting an existence of other mechanisms underlying anti-tumour effect in this model that are yet to be elucidated.


title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

abstract:
Early life SARS-CoV-2 vaccination has the potential to provide lifelong protection and achieve herd immunity. To evaluate SARS-CoV-2 infant vaccination, we immunized two groups of 8 infant rhesus macaques (RMs) at weeks 0 and 4 with stabilized prefusion SARS-CoV-2 S-2P spike (S) protein, either encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or mixed with 3M-052-SE, a TLR7/8 agonist in a squalene emulsion (Protein+3M-052-SE). Neither vaccine induced adverse effects. High magnitude S-binding IgG and neutralizing infectious dose 50 (ID50) >103 were elicited by both vaccines. S-specific T cell responses were dominated by IL-17, IFN-{gamma}, or TNF-. Antibody and cellular responses were stable through week 22. The S-2P mRNA-LNP and Protein-3M-052-SE vaccines are promising pediatric SARS-CoV-2 vaccine candidates to achieve durable protective immunity.

One-Sentence SummarySARS-CoV-2 vaccines are well-tolerated and highly immunogenic in infant rhesus macaques


title: CRISPRpas: Programmable regulation of alternative polyadenylation by dCas9

abstract: Well over half of human mRNA genes produce alternative polyadenylation (APA) isoforms that differ in mRNA metabolism due to 3 UTR size changes or have variable coding potentials when coupled with alternative splicing. Aberrant APA is implicated in a growing number of human diseases. A programmable tool for APA regulation, hence, would be instrumental for understanding how APA events impact biological processes. Here, using a catalytically dead Cas9 (dCas9), we developed a method, named CRISPRpas, to alter cleavage and polyadenylation site (PAS) usage in 3 UTRs or introns. We present key features that facilitate CRISPRpas, including targeting DNA strand, distance between PAS and targeting sequence, and strength of the PAS. For intronic PAS, we additionally analyze strengths of 5 splice site and target location in intron. Our analyses implicate a dynamic competition between PAS usage and nascent RNA decay when RNA polymerase II elongation is blocked. We show modulation of APA of multiple endogenous genes including a gene that contains a single nucleotide polymorphism (SNP) that affects APA in the human population. CRISPRpas expands the CRISPR toolkit for perturbation of gene expression.


title: Decreasing Mitochondrial RNA Polymerase Activity Reverses Biased Inheritance of Hypersuppressive mtDNA

abstract: Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that regions of rho+ mtDNA persisted after hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced hypersuppressive biased inheritance. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance relative to hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.


title: Early life Western diet-induced memory impairments and gut microbiome changes in female rats are long-lasting despite healthy dietary intervention

abstract:
ObjectiveConsumption of a Western diet during adolescence results in hippocampus (HPC)-dependent memory impairments and gut microbiome dysbiosis. Whether these adverse outcomes are reversible in adulthood following intervention with a healthy diet is unknown. Here we assessed the short- and long-term effects of adolescent consumption of a Western diet enriched with either sugar alone, or sugar and fat on metabolic outcomes, HPC-dependent memory, and gut microbiota.

MethodsAdolescent female rats (PN 26) were fed a standard chow diet (CTL), a chow diet with access to 11% sugar solution (SUG), or a junk food cafeteria-style diet (CAF) containing a variety of fat- and/or sugar-enriched foods. During adulthood (PN 65+), metabolic outcomes, HPC-dependent memory, and gut microbial populations were evaluated both before and after a 5-week dietary intervention period where all groups were fed a diet of water standard chow.

ResultsPrior to the dietary intervention both the CAF and SUG groups demonstrated impaired HPC-dependent memory, increased adiposity, and altered gut microbial populations relative to controls. However, impaired peripheral glucose regulation was only observed in the SUG group. The dietary intervention reversed the metabolic dysfunction in both the CAF and SUG groups, whereas HPC-dependent memory impairments were reversed in the SUG, but not the CAF group. The composition of the gut microbiota remained distinct from controls in both groups after dietary intervention.

ConclusionsWhile the metabolic impairments associated with adolescent cafeteria diet consumption are reversible in adulthood with dietary intervention, the HPC-dependent memory impairments and the gut microbiome dysbiosis persist.


title: Structure of the Bacteriophage PhiKZ non-Virion RNA Polymerase

abstract: Bacteriophage {Phi}KZ is the founding member of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. {Phi}KZ is an incredibly interesting virus, expressing many systems the host already possesses. On infection, it forms a "nucleus", erecting a barrier around its massive genome to exclude host restriction endonucleases and CRISPR-Cas systems. {Phi}KZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. {Phi}KZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial {beta}/{beta}' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of {Phi}KZ nvRNAP to 3.3 [A], shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, with structural homology and a biochemical role implying that it has evolved from an ancestral homologue to {sigma}-factor.


title: Ultrafast, alignment-free detection of repeat expansions in NGS and RNAseq data

abstract: Short tandem repeat expansions are an established cause of diseases such as Huntingtons disease. Bioinformatic methods for detecting repeat expansions in short-read sequencing have revealed new repeat expansions in humans. Current bioinformatic methods to detect repeat expansions require alignment information to identify repetitive motif enrichment at genomic locations. We present superSTR, an ultrafast method that does not require alignment. We demonstrate superSTRs ability to efficiently process both whole-genome and whole-exome sequencing data. Using superSTR we perform the first analysis of the UK Biobank to efficiently screen the exomes of 49,953 biobank participants for repeat expansions. We identify known mutations, as well as diseases not previously associated with REs. We further demonstrate the first bioinformatic screening of RNA sequencing data to detect repeat expansions in patients with spinocerebellar ataxia and Fuchs endothelial corneal dystrophy, and mouse models of myotonic dystrophy. superSTR is a highly computationally-efficient repeat expansion tool screening and detection tool for genomewide novel repeat expansion analysis, significantly outperforming existing methods. superSTR is available from https://github.com/bahlolab/superSTR.


title: Ultrafast, alignment-free detection of repeat expansions in next-generation DNA and RNA sequencing data


title: The Up state of the SARS-COV-2 Spike homotrimer favors an increased virulence for new variants

abstract:
The COVID-19 pandemic has spread widely worldwide. However, as soon as the vaccines were released - the only scientifically verified and efficient therapeutic option thus far - a few mutations combined into variants of SARS-CoV-2 that are more transmissible and virulent emerged raising doubts about their efficiency. Therefore, this work aims to explain possible molecular mechanisms responsible for the increased transmissibility and the increased rate of hospitalizations related to the new variants. A combination of theoretical methods was employed. Constant-pH Monte Carlo simulations were carried out to quantify the stability of several spike trimeric structures at different conformational states and the free energy of interactions between the receptor binding domain (RBD) and Angiotensin Converting Enzyme 2 (ACE2) for the most worrying variants. Electrostatic epitopes were mapped using the PROCEEDpKa method. These analyses showed that the increased virulence is more likely to be due to the improved stability to the S trimer in the opened state (the one in which the virus can interact with the cellular receptor ACE2) than due to alterations in the complexation RBD-ACE2, once the increased observed in the free energy values is small. Conversely, the South African variant (B.1.351), when compared with the wild type SARS-CoV-2, is much more stable in the opened state (either with one or two RBDs in the up position) than in the closed state (with the three RBDs in the down position). Such results contribute to the understanding of the natural history of disease and also to indicate possible strategies to both develop new therapeutic molecules and to adjust the vaccine doses for a higher production of B cells antibodies.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Triad interaction stabilizes the voltage sensor domains in a constitutively open KCNQ1-KCNE3 channel

abstract: Tetrameric voltage-gated K+ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K+ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the recently determined KCNQ1-KCNE3-calmodulin complex structure, we identified amino acid residues on KCNE3 facing the S1 segment of KCNQ1 that are required for constitutive activity. In addition, we found that the interaction of these amino acid residues of KCNE3 and the S1 segment affects the voltage sensor movement via M238 and V241 residues of the S4 segment. This triad interaction shifts the voltage sensor domains equilibrium, leading to stabilization of the channels open state.

category: biophysics


title: The barley mutant multiflorus2.b reveals quantitative genetic variation for new spikelet architecture

abstract:
Understanding the genetic basis of yield forming factors in small grain cereals is of extreme importance, especially in the wake of stagnation of further yield gains in these crops. One such yield forming factor in these cereals is the number of grain-bearing florets produced per spikelet. Wildtype barley (Hordeum vulgare L.) spikelets are determinate structures, the spikelet axis (rachilla) degenerates after producing single floret. In contrast, the rachilla of wheat (Triticum ssp.) spikelets, which are indeterminate, elongates to produce up to 12 florets. In our study, we characterized the barley spikelet determinacy mutant multiflorus2.b (mul2.b) that produced up to three fertile florets on elongated rachillae of lateral spikelets. Apart from the lateral spikelet indeterminacy (LS-IN), we also characterized the supernumerary spikelet phenotype in the central spikelets (CS-SS) of mul2.b. Through our phenotypic and genetic analyses, we identified two major QTLs on chromosomes 2H and 6H, and two minor QTLs on 3H for the LS-IN phenotype. For, the CS-SS phenotype we identified one major QTL on 6H, and a minor QTL on 5H chromosomes. Notably, the 6H QTLs for CS-SS and LS-IN phenotypes co-located with each other, potentially indicating that a single genetic factor might regulate both phenotypes. Thus, our in-depth phenotyping combined with genetic analyses revealed the quantitative nature of the LS-IN and CS-SS phenotypes in mul2.b, paving the way for cloning the genes underlying these QTLs in the future.

Key messageSpikelet indeterminacy and supernumerary spikelet phenotypes in barley multiflorus2.b mutant show polygenic inheritance. Genetic analysis of multiflorus2.b revealed major QTLs for spikelet determinacy and supernumerary spikelet phenotypes on 2H and 6H chromosomes.

category: plant biology


title: Synapse development is regulated by microglial THIK-1 K+ channels

abstract:
Microglia are the resident immune cells of the central nervous system. They constantly survey the brain parenchyma for redundant synapses, debris or dying cells, which they remove through phagocytosis. Microglial ramification, motility and cytokine release are regulated by tonically active THIK-1 K+ channels on the microglial plasma membrane. Here, we examined whether these channels play a role in phagocytosis. Using pharmacological blockers and THIK-1 knockout (KO) mice, we found that lack of THIK-1 activity reduced microglial phagocytosis, which may result in impaired pruning of synapses. In hippocampus, mice lacking THIK-1 expression had an increased number of glutamatergic synapses during development. This resulted from an increased number of presynaptic terminals, due to impaired removal by THIK-1 KO microglia. In microglia in brain slices from fresh human biopsies, modulating THIK-1 function had effects similar to those in rodents: blocking THIK-1 rapidly reduced microglial process ramification and increased synaptic density. The dependence of synapse number on THIK-1 K+ channels, which control microglial surveillance and phagocytic ability, implies that changes in THIK-1 expression level over the lifespan or in disease states may contribute to altering neural circuit function.

SignificanceMicroglia are the brains resident immune cells, surveying it with motile processes, which can remove pathogens but also prune unnecessary junctions between the neurons (synapses). A potassium channel, THIK-1, in the microglial membrane allows efflux of potassium from these cells, and thereby regulates their membrane voltage as well as their process motility and release of inflammatory mediators. Here, using THIK-1-blocking drugs and THIK-1-deficient mice, we demonstrate that THIK-1 controls removal of synaptic material by microglia, which reduces the number of functional synapses. We also show that blocking THIK-1, as some anaesthetics do, affects microglial structure and increases the number of synapses in living brain slices from both rodents and humans, and could thus alter network function in the brain.


title: Targeting properdin - Structure and function of a novel family of tick-derived complement inhibitors

abstract:
Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers.

We have identified a novel family of alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner.

For the first time this study provides a full functional and structural characterization of a properdin inhibitor, opening avenues for future therapeutic approaches.