title: Sub-Cluster Identification through Semi-SupervisedOptimization of Rare-cell Silhouettes (SCISSORS) in Single-Cell Sequencing

abstract: Single-cell RNA-sequencing (scRNA-seq) has enabled the molecular profiling of thousands to millions of cells simultaneously in biologically heterogenous samples. Currently, common practice in scRNA-seq is to determine cell type labels through unsupervised clustering and the examination of cluster-specific genes. However, even small differences in analysis and parameter choice can greatly alter clustering solutions and thus impose great influence on which cell types are identified. Existing methods largely focus on determining the optimal number of robust clusters, which is not favorable for identifying cells of extremely low abundance due to their subtle contributions towards overall patterns of gene expression. Here we present a carefully designed framework, SCISSORS, which accurately profiles subclusters within major cluster(s) for the identification of rare cell types in scRNA-seq data. SCISSORS employs silhouette scoring for the estimation of heterogeneity of clusters and reveals rare cells in heterogenous clusters by implementing a multi-step, semi-supervised reclustering process. Additionally, SCISSORS provides a method for the identification of marker genes of rare cells, which may be used for further study. SCISSORS is wrapped around the popular Seurat R package and can be easily integrated into existing Seurat pipelines. SCISSORS, including source code and vignettes for two example datasets, is freely available at https://github.com/jrleary/SCISSORS.

category: bioinformatics


title: Allosteric modulation of the SARS-CoV-2 spike conformation

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Forster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

category: biophysics


title: Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike


title: Improving mathematical models of cancer by including resistance to therapy: a study in non-small cell lung cancer

abstract: In this paper, a large dataset of 590 Non-Small Cell Lung Patients treated with either chemotherapy or immunotherapy was used to determine whether a game-theoretic model including both evolution of therapy resistance and cost of resistance provides a better fit than classical mathematical models of population growth (exponential, logistic, classic Bertalanffy, general Bertalanffy, Gompertz, general Gompertz). This is the first time a large clinical patient cohort (as opposed to only in-vitro data) has been used to apply a game-theoretic cancer model. The game-theoretic model provides a better fit to the tumor dynamics of the 590 Non-Small Cell Lung Cancer patients than any of the non-evolutionary population growth models. This is not simply due to having more parameters in the game-theoretic model. The game-theoretic model is able to fit accurately patients whose tumor burden exhibit a U-shaped trajectory over time. We then demonstrate how this game-theoretic model provides predictions of tumor growth based on just a few initial measurements. Assuming that treatment-specific parameters define the treatment impact completely, we then explore alternative treatment protocols and their impact on the tumor growth. As such, the model can be used to suggest patient-specific optimal treatment regimens with the goal of minimizing final tumor burden. Therapeutic protocols based on game-theoretic modeling can predict tumor growth, and improve patient outcome. The model invites evolutionary therapies that anticipate and steer the evolution of therapy resistance.

category: cancer biology


title: Dry Powders for Inhalation Containing Monoclonal Antibodies Made by Thin-Film Freeze-Drying

abstract: Thin-film freeze-drying (TFFD) is a rapid freezing and then drying technique used to prepare inhalable dry powders from the liquid form for drug delivery to the lungs. We report the preparation of aerosolizable dry powders of monoclonal antibodies (mAbs) by TFFD. We first formulated IgG with lactose/leucine (60:40 w/w) or trehalose/leucine (75:25). IgG 1% (w/w) formulated with lactose/leucine (60:40 w/w) in phosphate buffered saline (PBS) (IgG-1-LL-PBS) and processed by TFFD was found to produce the powder with the most desirable aerosol properties. We then replaced IgG with a specific antibody, anti-programmed cell death protein (anti-PD-1 mAb), to prepare a dry powder (anti-PD1-1-LL-PBS), which performed similarly to the IgG-1-LL-PBS powder. The aerosol properties of anti-PD1-1-LL-PBS were significantly better when TFFD was used to prepare the powder as compared to conventional shelf freeze-drying (shelf FD). The dry powder had a porous structure with nanoaggregates. The dry powder had a Tg value between 39-50 {degrees}C. When stored at room temperature, the anti-PD-1 mAb in the TFFD powder was more stable than that of the same formulation stored as a liquid. The addition of polyvinylpyrrolidone (PVP) K40 in the formulation was able to raise the Tg to 152 {degrees}C, which is expected to further increase the storage stability of the mAbs. The PD-1 binding activities of the anti-PD-1 mAbs before and after TFFD were not different. While protein loss, likely due to protein binding to glass or plastic vials and the TFF apparatus, was identified, we were able to minimize the loss by increasing mAb content in the powders. Lastly, we show that another mAb, anti-TNF-, can also be converted to a dry powder with a similar composition by TFFD. We conclude that TFFD can be applied to produce stable aerosolizable dry powders of mAbs for pulmonary delivery.

category: pharmacology and toxicology


title: Metabolic evidence for distinct pyruvate pools inside plant mitochondria

abstract:
The majority of the pyruvate inside plant mitochondria is either transported into the matrix from the cytosol via the mitochondria pyruvate carrier (MPC) or synthesised in the matrix by alanine aminotransferase (AlaAT) or NAD-malic enzyme (NAD-ME). Pyruvate from these origins could mix into a single pool in the matrix and contribute indistinguishably to respiration, or they could maintain a degree of independence in metabolic regulation. Here, we demonstrated that feeding isolated mitochondria with U-13C-pyruvate and unlabelled malate enables the assessment of pyruvate contribution from different sources to TCA cycle intermediate production. Imported pyruvate is the preferred source for citrate production even when the synthesis of NAD-ME-derived pyruvate was optimised. Genetic or pharmacological elimination of MPC activity removed this preference and allowed an equivalent amount of citrate to be generated from the pyruvate produced by NAD-ME. Increasing mitochondrial pyruvate pool size by exogenous addition only affected metabolites from pyruvate transported by MPC whereas depleting pyruvate pool size by transamination to alanine only affected metabolic products derived from NAD-ME. Together, these data reveal respiratory substrate supply in plants involves distinct pyruvate pools inside the matrix that can be flexibly mixed based on the rate of pyruvate transport from the cytosol. These pools are independently regulated and contribute differentially to organic acids export from plant mitochondria.

Significance statementPyruvate is the primary respiratory substrate for energy production to support plant growth and development. However, it is also the starting material of many other pathways. Prioritisation of respiratory use over other competing pathways would enable a level of control when pyruvate is delivered to mitochondria via the mitochondrial pyruvate transporter. We demonstrated the existence of two distinct pyruvate pools in plant mitochondria suggesting inner mitochondrial organisation allows metabolic heterogeneity, hence metabolic specialisation. This explains why NAD-ME flux into plant respiration is low and confirms the prominent link between imported pyruvate and energy production. This compartmentation also reveals how NAD-ME supplies substrate to the mitochondrial pyruvate exporter in plants, especially during C4 metabolism.

category: plant biology


title: Suppression of a dsRNA-induced plant immunity pathway by viral movement protein

abstract: The virome of plants is dominated by RNA viruses 1 and several of these cause devastating diseases in cultivated plants leading to global crop losses 2. To infect plants, RNA viruses engage in complex interactions with compatible plant hosts. In cells at the spreading infection front, RNA viruses replicate their genome through double-stranded RNA (dsRNA) intermediates and interact with cellular transport processes to achieve cell-to-cell movement of replicated genome copies through cell wall channels called plasmodesmata (PD) 3. In order to propagate, viruses also must overcome host defense responses. In addition to triggering the antiviral RNA silencing response, RNA virus infection also elicits pattern-triggered immunity (PTI) 4 whereby dsRNA, a hallmark of virus replication, acts as an important elicitor 5. This innate antiviral immune response is also triggered when dsRNA is applied externally and does not require sequence homology to the virus 5. However, the mechanism by which PTI restricts virus infection is not known. Here, we show that dsRNA inhibits the progression of virus infection by triggering callose deposition at plasmodesmata and the inhibition of transport through these cell-to-cell communication channels. The dsRNA-induced signaling pathway leading to callose deposition is independent of ROS production and thus distinguished from pathways triggered by bacterial and fungal elicitors. The dsRNA-induced host response at plasmodesmata is suppressed by the Tobacco mosaic virus movement protein (MP). Thus, the virus uses MP to inhibit innate dsRNA-induced immunity at plasmodesmata, which could be a general strategy of phytoviruses to overcome plant defenses and spread infection.


title: A temperature sensitive mutation in the CstF77 subunit of the polyadenylation complex reveals the critical function of mRNA 3' end formation for a robust heat stress response in plants

abstract:
BackgroundHeat Shock Protein 101 (HSP101) in plants and orthologs in bacteria (Caseinolytic peptidase B, ClpB) and yeast (Hsp104) are essential for thermotolerance. To investigate molecular mechanisms of thermotolerance involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a semi-dominant, missense HSP101 allele, hot1-4 (A499T). Plants carrying the hot1-4 mutation are more heat-sensitive than an HSP101 null mutant (hot1-3), indicating the toxicity of hot1-4 allele.

ResultsWe report that one suppressor (shot2, suppressor of hot1-4 2) has a temperature-sensitive, missense mutation (E170K) in the CstF77 (Cleavage stimulation factor 77) subunit of the polyadenylation complex, which is critical for 3 end maturation of pre-mRNA. RNA-Seq analysis of total RNA depleted of ribosomes reveals that heat treatment causes transcriptional readthrough events in shot2, specifically in highly heat-induced genes, including the toxic hot1-4 gene. In addition, failure of correct transcript processing leads to reduced accumulation of many HSP RNAs and proteins, suppressing heat sensitivity of the hot1-4 mutant, due to reduction of the toxic mutant HSP101 protein. Notably, the shot2 mutation makes plants more sensitive to heat stress in the HSP101 null (hot1-3) and wild-type backgrounds correlated with the reduced expression of other heat-inducible genes required for thermotolerance.

ConclusionsOur study reveals the critical function of CstF77 for 3 end formation of mRNA during heat stress, as well as the dominant role of HSP101 in dictating the outcome of severe heat stress in plants.


title: Conserved and newly acquired roles of PIF1 homologs in tomato (Solanum lycopersicum)

abstract: PHYTOCHROME INTERACTING FACTORS (PIFs) are transcription factors that interact with the photoreceptors phytochromes and integrate multiple signaling pathways related to light, temperature, defense and hormone responses. PIFs have been extensively studied in Arabidopsis thaliana, but less is known about their roles in other species. Here, we investigate the role of the two homologs of PIF1 found in tomato (Solanum lycopersicum), namely PIF1a and PIF1b. Analysis of gene expression showed very different patterns, indicating a potential evolutionary divergence in their roles. At the protein level, light regulated the stability of PIF1a, but not PIF1b, further supporting a functional divergence. Phenotypic analyses of CRISPR-Cas9-generated tomato mutants defective in PIF1a or PIF1b or both revealed conserved and newly acquired roles compared to Arabidopsis PIF1. Both PIF1a or PIF1b were found to regulate seed germination, photosynthetic pigment biosynthesis and fruit production. However, only PIF1a-defective mutants showed defects on root hair elongation, flowering time and fruit growth and softening. We did not identify any process altered only in plants lacking PIF1b. Together, these data show that neofunctionalization has taken place in tomato, illustrating the potential of these transcription factors to acquiring new roles in different species.


title: Selective Control of Synaptically-Connected Circuit Elements by All-Optical Synapses

abstract: Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time  Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic  Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control during behavior in vivo.

category: neuroscience


title: Tracking neural activity from the same cells during the entire adult life of mice

abstract: Recording the activity of the same neurons over the adult life of an animal is important to neuroscience research and biomedical applications. Current implantable devices cannot provide stable recording on this time scale. Here, we introduce a method to precisely implant nanoelectronics with an open, unfolded mesh structure across multiple brain regions in the mouse. The open mesh structure forms a stable interwoven structure with the neural network, preventing probe drifting and showing no immune response and neuron loss during the yearlong implantation. Using the implanted nanoelectronics, we can track single-unit action potentials from the same neurons over the entire adult life of mice. Leveraging the stable recordings, we build machine learning algorithms that enable automated spike sorting, noise rejection, stability validation, and generate pseudotime analysis, revealing aging-associated evolution of the single-neuron activities.


title: Blast-induced mild traumatic brain injury alterations of corticotropin-releasing factor neuronal activity in the mouse hypothalamic paraventricular nucleus

abstract: Blast-induced mild traumatic brain injury (mbTBI) is the most common cause of TBI in US service members and veterans. Those exposed to TBI are at greater risk of developing neuropsychiatric disorders such as posttraumatic stress disorder, anxiety and depressive disorders, and substance use disorders following TBI [1, 2]. Previously, we have demonstrated that mbTBI increases anxiety-like behaviors in mice and dysregulates the stress at the level of corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus (PVN). To expand on how mTBI may dysregulate the stress axis centrally, here PVN CRF neuronal activity was evaluated using whole cell-patch clamp recordings in hypothalamic slices from sham and mbTBI adult male CRF:tdTomato mice 7 days post-injury. We found that mbTBI generally did not affect the neuronal excitability and intrinsic membrane properties of PVN CRF neurons; this injury selectively increased the frequency of spontaneous neuronal firing of PVN CRF neurons localized to the dorsal PVN (dPVN) but not ventral PVN (vPVN). Consistently, mbTBI-induced dPVN CRF hyperactivity was associated with pre- and post-synaptic depression of spontaneous GABAergic transmission onto dPVN CRF neurons suggesting that mbTBI-induced GABAergic synaptic dysfunction may underlie dPVN CRF neuronal hyperactivity and increases in dPVN CRF signaling. The present results provide the first evidence for mbTBI-induced alterations in PVN CRF neuronal activity and GABAergic synaptic function that could mediate hypothalamic CRF dysregulation following mbTBI contributing to stress psychopathology associated with blast injury.


title: Serial invasions can disrupt the time course of ecosystem recovery

abstract: The impacts of species invasions can subside or amplify over time as ecosystems "adapt" or additional invaders arrive. These long-term changes provide important insights into ecosystem dynamics. Yet studies of long-term dynamics are rare and often confound species impacts with coincident environmental change. We synthesize many-decade time-series across ecosystems to resolve shared changes in seven key features following invasion by quagga and zebra mussels, two widespread congeners that re-engineer and increasingly co-invade freshwaters. Six polymictic shallow lakes with long-term data sets reveal remarkably similar trends, with the strongest ecosystem impacts occurring within 5-10 years of zebra mussel invasion. Surprisingly, plankton communities then exhibited a partial, significant recovery. This recovery was absent, and impacts of initial invasion amplified, in lakes where quagga mussels outcompeted zebra mussels and more completely depleted phytoplankton. Thus, invasion impacts subside over time but can amplify with serial introductions of competing, even closely similar, taxa.

category: ecology


title: Analyzing and Reconciling Colocalization and Transcriptome-wide Association Studies from the Perspective of Inferential Reproducibility

abstract: Transcriptome-wide association studies and colocalization analysis are popular computational approaches for integrating genetic association data from molecular and complex traits. They show the unique ability to go beyond variant-level genetic association evidence and implicate critical functional units, e.g., genes, in disease etiology. However, in practice, when the two approaches are applied to the same molecular and complex trait data, the inference results can be markedly different. This paper systematically investigates the inferential reproducibility between the two approaches through theoretical derivation, numerical experiments, and analyses of 4 complex trait GWAS and GTEx eQTL data. We identify two classes of inconsistent inference results. We find that the first class of inconsistent results may suggest an interesting biological phenomenon, i.e., horizontal pleiotropy; thus, the two approaches are truly complementary. The inconsistency in the second class can be understood and effectively reconciled. To this end, we propose a novel approach for locus-level colocalization analysis. We demonstrate that the joint TWAS and locus-level colocalization analysis improves specificity and sensitivity for implicating biological-relevant genes.

category: genetics


title: Mouse Organ Specific Proteins and Functions

abstract: Organ specific proteins (OSPs) possess great medical potentials both in clinics and in biomedical research. Applications of them -- such as alanine transaminase, aspartate transaminase, and troponins -- in clinics have raised certain concerns of their organ specificity. The dynamics and diversity of protein expression in heterogeneous human population are well known, yet their effects on OSPs are less addressed. Here we use mouse as a model and implemented a scheme of breadth study to examine the pan-organ proteome for potential variations of organ specificity in different genetic backgrounds. Using reasonable resources, we generated pan-organ proteomes of four in-bred mouse strains. The results revealed a large diversity that is more profound among OSPs than the overall proteomes. We defined a robustness score to quantify such variation and derived three sets of OSPs with different stringencies. In the meantime, we found that the enriched biological functions of OSPs are also organ specific that are sensitive and useful to assess the quality of OSPs. We hope our breadth study can open doors to explore the molecular diversity and dynamics of organ specificity at the protein level.

category: genomics


title: Reversal of lineage plasticity in RB1/TP53-deleted prostate cancer through FGFR and Janus kinase inhibition

abstract: The inherent plasticity of tumor cells provides a mechanism of resistance to many molecularly targeted therapies, exemplified by adeno-to-neuroendocrine lineage transitions seen in prostate and lung cancer. Here we investigate the root cause of this lineage plasticity in a primary murine prostate organoid model that mirrors the lineage transition seen in patients. These cells lose luminal identity within weeks following deletion of Trp53 and Rb1, ultimately acquiring an Ar-negative, Syp+ phenotype after orthotopic in vivo transplantation. Single-cell transcriptomic analysis revealed progressive mixing of luminal-basal lineage features after tumor suppressor gene deletion, accompanied by activation of Jak/Stat and Fgfr pathway signaling and interferon-a and -g gene expression programs prior to any morphologic changes. Genetic or pharmacologic inhibition of Jak1/2 in combination with FGFR blockade restored luminal differentiation and sensitivity to antiandrogen therapy in models with residual AR expression. Collectively, we show lineage plasticity initiates quickly as a largely cell-autonomous process and, through newly developed computational approaches, identify a pharmacological strategy that restores lineage identity using clinical grade inhibitors.


title: Visceral Fat Inflammation and Fat Embolism are associated with Lung's Lipidic Hyaline Membranes in COVID-19 patients

abstract:
BackgroundVisceral obesity is a critical determinant of severe coronavirus disease-2019 (COVID-19). Methods: In this study, we performed a comprehensive histomorphologic analysis of autoptic visceral adipose tissues (VAT), lungs and livers of 19 COVID-19 and 23 non-COVID-19 subjects.

ResultsAlthough there were no between-groups differences in body-mass-index and adipocytes size, higher prevalence of CD68+ macrophages in COVID-19 subjects VAT was detected (p=0.005) and accompanied by crown-like structures presence, signs of adipocytes stress and death. Consistently, human adipocytes were successfully infected by SARS-CoV2 in vitro and displayed lower cell viability. Being VAT inflammation associated with lipids spill-over from dead adipocytes, we studied lipids distribution employing Oil-Red-O staining (ORO). Lipids were observed within lungs and livers interstitial spaces, macrophages, endothelial cells, and vessels lumen, features suggestive of fat embolism syndrome, more prevalent among COVID-19 individuals (p<0.001). Notably, signs of fat embolism were more prevalent among obese (p=0.03) independently of COVID-19 diagnosis, suggesting that such condition may be an obesity complication, exacerbated by SARS-CoV2 infection. Importantly, all infected subjects lungs presented lipids-rich (ORO+) hyaline membranes, formations associated with COVID-19-related pneumonia, present only in one control with non-COVID-19 pneumonia.

ConclusionsThis study describes for the first time novel COVID-19-related features possibly underlying the unfavorable prognosis in obese SARS-CoV2-infected-subjects.

category: pathology


title: The mitochondrial gene CMPK2 functions as a rheostat for macrophage homeostasis in inflammation

abstract: In addition to their role in cellular energy production, mitochondria are increasingly recognized as regulators of the innate immune response of phagocytes. Here, we demonstrate that altering expression levels of the mitochondria associated enzyme, cytidine monophosphate kinase 2 (CMPK2), disrupts mitochondrial physiology and significantly de-regulates the resting immune homeostasis of macrophages. Both CMPK2 silenced as well as constitutively over expressing macrophage lines portray mitochondrial stress with marked depolarization of their membrane potential, enhanced ROS and disturbed architecture culminating in the enhanced expression of the pro-inflammatory genes-IL1{beta}, TNF and IL8. Interestingly, the long-term modulation of CMPK2 expression resulted in an increased glycolytic flux of macrophages akin to the altered physiological state of activated M1 macrophages. While infection induced inflammation for restricting pathogens is regulated, our observation of a total dysregulation of basal inflammation by bidirectional alteration of CMPK2 expression, only highlights a critical role of this gene in mitochondria mediated control of inflammation.

category: immunology


title: An atlas of human viruses provides new insights into diversity and tissue tropism of human viruses

abstract: Viruses continue to threaten human health. Yet, the complete viral species carried by humans and their infection characteristics have not been fully revealed. This study curated an atlas of human viruses from public databases and literatures, and built the Human Virus Database (HVD) available at http://computationalbiology.cn/humanVirusBase/#/. The HVD contains 1,131 virus species of 54 viral families which were more than twice the number of the human-infecting virus species reported in previous studies. These viruses were identified in human samples including 68 human tissues, the excreta and body fluid. The viral diversity in humans was age-dependent with a peak in the infant and a valley in the teenager. The tissue range of viruses was found to be associated with several factors including the viral group (DNA, RNA or reverse-transcribing viruses), enveloped or not, viral genome length and GC content, viral receptors and the virus-interacting proteins. Finally, the tissue range of DNA viruses was predicted using a random-forest algorithm with a medium performance. Overall, the study not only provides a valuable resource for further studies of human viruses, but also deepens our understanding towards the diversity and tissue tropism of human viruses.

category: microbiology


title: Polyamines and eIF5A hypusination facilitate SREBP2 translation and cholesterol synthesis to enhance enterovirus attachment and infection

abstract: Metabolism is key to cellular processes that underlie the ability of a virus to productively infect. Polyamines are small metabolites vital for many host cell processes including proliferation, transcription, and translation. Polyamine depletion also inhibits virus infection via diverse mechanisms, including inhibiting polymerase activity and viral translation. We showed that Coxsackievirus B3 (CVB3) attachment requires polyamines; however, the mechanism was unknown. Here, we report polyamines involvement in translation, through a process called hypusination, promotes expression of cholesterol synthesis genes by supporting SREBP2 translation, the master transcriptional regulator of cholesterol synthesis genes. Measuring bulk transcription, we found polyamines support expression of cholesterol synthesis genes, regulated by SREBP2. Polyamine depletion inhibits CVB3 by depleting cellular cholesterol. Exogenous cholesterol rescues CVB3 attachment, and mutant CVB3 resistant to polyamine depletion exhibits resistance to cholesterol perturbation. This study provides a novel link between polyamine and cholesterol homeostasis, a mechanism through which polyamines impact CVB3 infection.


title: Assessment of Plasmodium falciparum infection and fitness of genetically modified Anopheles gambiae aimed at mosquito population replacement

abstract: Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory Anopheles gambiae colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to Plasmodium falciparum NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both P. falciparum sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito population replacement, although the impact of its reduced fitness on the spread of the transgene through wild populations requires further investigation.


title: Yersinia pseudotuberculosis doxycycline tolerance strategies include modulating expression of genes involved in cell permeability and tRNA modifications

abstract: Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, may play an important role in promoting tolerance. We believe this is the first observation of bactericidal activity of doxycycline, which was revealed by reversing tusB downregulation.


title: Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in PSC.

abstract:
ObjectiveConflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. Goal: Define complex interactions between resident microbes and their association in PSC patients by studying antibiotic-treated specific pathogen-free (SPF) and germ-free (GF) multi-drug-resistant 2 deficient (mdr2-/-) mice.

DesignWe measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, fecal 16s rRNA gene profiling, and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients.

ResultsGF mdr2-/- mice had exaggerated hepatic inflammation and fibrosis with 100% mortality by 8 weeks; early SPF autologous stool transplantation rescued liver-related mortality. Broad-spectrum antibiotics and vancomycin alone accelerated disease in weanling SPF mdr2-/- mice, indicating that vancomycin-sensitive resident microbiota protect against hepatobiliary disease. Vancomycin treatment selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased cytolysin-expressing E. faecalis and E. coli liver translocation; colonization of gnotobiotic mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated liver inflammation and mortality. Lachnospiraceae colonization of antibiotic pre-treated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, while Lachnospiraceae-produced SCFA decreased fibrosis. Fecal E. faecalis/ Enterobacteriaceae was positively and Lachnospiraceae was negatively associated with PSC patients clinical severity Mayo risk scores.

ConclusionsWe identified specific functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical outcomes. These insights may guide personalized targeted therapeutic interventions in PSC patients.


title: Multiple spillovers and onward transmission of SARS-Cov-2 in free-living and captive White-tailed deer (Odocoileus virginianus)

abstract:
Many animal species are susceptible to SARS-CoV-2 and could potentially act as reservoirs, yet transmission of the virus in non-human free-living animals has not been documented. White-tailed deer (Odocoileus virginianus), the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 may be circulating in deer, we tested 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through December of 2020 for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 deer (33.2%; 95% CI: 28, 38.9) samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, between November 23, 2020 and January 10, 2021, 80 of 97 (82.5%; 95% CI 73.7, 88.8) RPLN samples had detectable SARS-CoV-2 RNA by RT-PCR. Whole genome sequencing of the 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%), and B.1.311 (n = 19; 20%) accounting for ~75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple zooanthroponotic spillover events and deer-to-deer transmission. The discovery of sylvatic and enzootic SARS-CoV-2 transmission in deer has important implications for the ecology and long-term persistence, as well as the potential for spillover to other animals and spillback into humans. These findings highlight an urgent need for a robust and proactive "One Health" approach to obtaining a better understanding of the ecology and evolution of SARS-CoV-2.

One-Sentence SummarySARS-CoV-2 was detected in one-third of sampled white-tailed deer in Iowa between September 2020 and January of 2021 that likely resulted from multiple human-to-deer spillover and deer-to-deer transmission events.


title: Multiple spillovers and onward transmission of SARS-Cov-2 in free-living and captive White-tailed deer


title: A biosafety level 2 surrogate for studying SARS-CoV-2 survival in food processing environmental biofilms

abstract: Meat processing plants have been at the center of the SARS-CoV-2 pandemic. There are several factors that contribute to the persistence of SARS-CoV-2 in meat processing plants and one of the factors is the formation of a multi-species biofilm with virus. Biofilm can act as a reservoir in protecting, harboring, and dispersing SARS-CoV-2 from biofilm to the meat processing facility environment. We used Murine Hepatitis Virus (MHV) as a surrogate for SARS-CoV-2 virus and meat processing facility drain samples to develop mixed-species biofilms on commonly found materials in processing facilities (Stainless-Steel (SS), PVC and tiles). The results showed that MHV was able to integrate into the environmental biofilm and survived for a period of 5 days at 7{degrees}C. There was no significate difference between the viral-environmental biofilm biovolumes developed on different materials SS, PVC, and tiles. There was a 2-fold increase in the virus-environmental biofilm biovolume when compared to environmental biofilm by itself. These results indicate a complex virus-environmental biofilm interaction which is providing enhanced protection for the survival of viral particles with the environmental biofilm community.


title: Distance-dependent inhibition of translation initiation by downstream out-of-frame AUGs reveals that ribosome scans in a Brownian ratchet process

abstract: While eukaryotic ribosomes are widely presumed to scan mRNA for the AUG codon to initiate translation in a strictly 5'-3' movement (strictly unidirectional scanning model), other evidence has suggested that the ribosome uses small-amplitude 5'-3' and 3'-5' oscillations with a net 5'-3' movement to recognize the AUG codon (Brownian ratchet scanning model). Here, we generated 13,437 yeast variants, each with an ATG triplet placed downstream (dATGs) of the annotated ATG (aATG) codon of green fluorescent protein. We found that out-of-frame dATGs could inhibit translation at the aATG, but with diminishing strength over increasing distance between aATG and dATG, undetectable beyond [~]17 nt. Computational simulations revealed that each triplet is scanned back and forth approximately [~]10 times until an AUG codon is recognized. Collectively, our findings uncover the basic process by which eukaryotic ribosomes scan for initiation codons, and how this process could shape eukaryotic genome evolution and influence cancer development.

category: molecular biology


title: Quantitative Measurement of Diffusion-weighted Imaging Signal Using Expression-controlled Aquaporin-4 Cells: Comparative Study of 2-compartment and Diffusion Kurtosis Imaging Models

abstract:
PurposeThe purpose of this study was to compare parameter estimates for the 2-compartment (2Comp) and diffusion kurtosis imaging (DKm) models obtained from diffusion-weighted imaging (DWI) of aquaporin-4 (AQP4) expression-controlled cells, and to look for biomarkers that indicate differences in the cell membrane water permeability.

MethodsDWI was performed on AQP4-expressing and non-expressing cells and the signal was analyzed with the 2Comp and DKm models. For the 2Comp model, the diffusion coefficients (Df, Ds) and volume fractions (Ff, Fs, Ff=1-Fs) of the fast and slow compartments were estimated. For the DKm model, estimates of the diffusion kurtosis (K) and corrected diffusion coefficient (D) were obtained.

ResultsFor the 2Comp model, Ds and Fs showed clear differences between AQP4-expressing and non-expressing cells. Fs was also sensitive to cell density. There was no clear relationship with the cell type for the DKm parameters.

ConclusionsChanges to cell membrane water permeability due to AQP4 expression affected DWI of cell suspensions. For the 2Comp and DKm models, Ds was the parameter most sensitive to differences in AQP4 expression.

category: cell biology


title: Lack of evidence for a role of anthrax toxin receptors as surface receptors for collagen VI and for its cleaved off C5 domain (endotrophin)

abstract: The widely expressed microfibril-forming collagen VI is subject to proteolytic cleavage and it has been proposed that the cleaved off C-terminal Kunitz domain (C5) of the 3 chain is an adipokine important for tumor progression and fibrosis. Under the name "endotrophin" the C5 fragment has also been shown to be a potent biomarker for fibro-inflammatory diseases. However, the biochemical mechanisms behind endotrophin activity have not been investigated. In earlier studies, the anthrax toxin receptor 1 was found to bind to C5, but this potential interaction has not been further studied. Given the proposed physiological role of endotrophin we aimed to determine how the endotrophin signal is transmitted to the recipient cells. Surprisingly, we could not detect any interaction between endotrophin and anthrax toxin receptor 1 or its close relative, anthrax toxin receptor 2. Moreover, we could not detect binding of fully assembled collagen VI to either anthrax toxin receptor. We also performed similar experiments with the collagen VI surface receptor NG2 (CSPG4). We could confirm that NG2 is a collagen VI receptor that binds to assembled collagen VI, but not to the cleaved C5/endotrophin. A cellular receptor for C5/endotrophin therefore still remains elusive.


title: Spatial dynamics and vaccine-induced fitness changes of Bordetella pertussis

abstract: Competitive interactions between pathogen strains drive infection risk. Vaccines are thought to perturb strain diversity through shifts in immune pressures, however, this has rarely been measured due to inadequate data and analytical tools. Bordetella pertussis (B. pertussis), responsible for 160,000 deaths annually1, provides a rare natural experiment as many countries have switched from whole cell vaccines to acellular vaccines, which have very different immunogenic properties2,3. Here we use 3,344 sequences from 23 countries and build phylogenetic models to reveal that B. pertussis has substantial diversity within communities, with the relative fitness of local genotypes changing in response to switches in vaccine policy. We demonstrate that the number of transmission chains circulating within subnational regions is strongly associated with host population size. It takes 5-10 years for individual lineages to be homogeneously distributed throughout Europe or the United States. Increased fitness of pertactin-deficient strains following implementation of acellular vaccines, but reduced fitness otherwise, can explain long-term genotype dynamics. These findings highlight the role of national vaccine policies in shifting local diversity of a pathogen that still poses a large burden on global public health.


title: Genomic analysis reveals geography rather than culture as the predominant factor shaping genetic variation in northern Kenyan human populations

abstract:
ObjectivesThe aim of this study was to characterize the genetic relationships within and among four neighboring populations in northern Kenya in light of cultural relationships to understand the extent to which geography and culture shape patterns of genetic variation.

Materials and MethodsWe collected DNA and demographic information pertaining to aspects of social identity and heritage from 572 individuals across the Turkana, Samburu, Waso Borana, and Rendille of northern Kenya. We sampled individuals across a total of nine clans from these four groups and, additionally, three territorial sections within the Turkana and successfully genotyped 376 individuals.

ResultsHere we report that geography predominately shapes genetic variation within and among human groups in northern Kenya. We observed a clinal pattern of genetic variation that mirrors the overall geographic distribution of the individuals we sampled. We also found relatively higher rates of intermarriage between the Rendille and Samburu and evidence of gene flow between them that reflect these higher rates of intermarriage. Among the Turkana, we observed strong recent genetic substructuring based on territorial section affiliation. Within ethnolinguistic groups, we found that Y chromosome haplotypes do not consistently cluster by natal clan affiliation. Finally, we found that sampled populations that are geographically closer have lower genetic differentiation, and that cultural similarity does not predict genetic similarity as a whole across these northern Kenyan populations.

DiscussionOverall, the results from this study highlight the importance of geography, even on a local geographic scale, in shaping observed patterns of genetic variation in human populations.


title: Mitonuclear interactions affect locomotor activity and sleep in Drosophila melanogaster

abstract: Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved between many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. The results demonstrate that Drosophila strains from different locations differ in sleep and activity, and the extent of variation differs between sexes, females in general being more active. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we establish that mtDNA variation affects both traits, sex specifically. Furthermore, by using previously published mtDNA copy number data, we detected a positive correlation between mtDNA copy number and the activity levels of the cybrid flies. Altogether, our study shows that both mtDNA variation and mitonuclear interactions affect activity and sleep patterns, highlighting the important role that both genomes play on life-history trait evolution.


title: Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

abstract:
Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as [~]36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.

One Sentence SummaryA new technology combining CRISPR-mediated genome editing and site-specific integrases enables efficient programmable gene integration at any targeted genomic locus without double-strand DNA breaks, leading to broad applications in basic science research, cell engineering, and gene therapy.

category: bioengineering


title: Air plasma-activated medium exerts tumor-specific cytotoxicity via the oxidative stress-induced perinuclear mitochondrial clustering

abstract:
Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy eliminating cancers effectively and safely is the current clinical choice. Since aggressive tumors have inherent or acquired resistance to multidisciplinary therapies targeting apoptosis, tumor-specific induction of another cell death modality is a promising avenue to meet the goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can induce cell death in OS and OC via a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) because of the characteristic unipolar mitochondrial perinuclear aggregation. APAM had potent antitumor activity both in vitro and in vivo. APAM caused apoptosis, necrotic cell death, and autophagy. APAM contained hydrogen peroxide and increased mitochondrial ROS (mROS), while the antioxidant N-acetylcysteine (NAC) prevented cell death. MPMC occurred following mitochondrial fragmentation coinciding with nuclear damages. MPMC was accompanied by the tubulin network remodeling and mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC and the microtubule inhibitor, Nocodazole. Increased Cardiolipin (CL) oxidation was also seen, and NAC and the peroxy radical scavenger Ferrostatin-1 prevented it. In contrast, in fibroblasts, APAM induced minimal cell death, mROS generation, mLPO accumulation, CL oxidation, and MPMC. These results suggest that MPMC is a tumor-specific cause of cell death via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC might serve as a promising target for exerting tumor-specific cytotoxicity.

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title: Improving the Virtual Trichrome Assessment through Bridge Category Models

abstract: Non-alcoholic steatohepatitis (NASH) is a liver disease characterized by excessive lipid accumulation and disease progression is typically assessed through inspection of a Trichrome stain for Fibrosis staging. As the public health burden of NASH worsens due to evolving lifestyle habits, pathology laboratory resources will become increasingly strained due to rising demand for specialized stains. Virtual staining processes, computational methods which can synthesize the application of chemical staining reagents, can potentially provide resource savings by obviating the need to acquire specialized stains. Virtual staining technologies are assessed by comparing virtual and real tissue stains for their realism and ability to stage. However, these assessment methods are rife with statistical mistreatment of observed phenomena that are difficult to account for. Bridge category ratings represent a phenomenon where a pathologist may assign two adjacent stages simultaneously, which may bias and/or reduce the power of research findings. Such stage assignments were frequently reported in a large-scale assessment of Virtual Trichrome technologies yet were unaccounted for since no statistical adjustment procedures existed. In this work, we provide an updated assessment of Virtual Trichrome technologies using Bridge Category Models, which account for these bridge ratings. We report that two of four pathologists tended to assign lower Fibrosis stages to virtually stained tissue while the other two pathologists assigned similar stages. These research findings differ when bridge ratings are not accounted for. While promising, these results indicate further room for algorithmic finetuning of Virtual Trichrome technologies.


title: Identification of marker-trait associated SNPs for fruit weight and plant height in a wild and domesticated jujube fruit tree (Ziziphus spp.) collection using genotyping-by-sequencing

abstract: Ziziphus are economically and nutritionally important fruiting plants that were domesticated in China around 7000 years ago. We identified genetic diversity in 141 accessions of four, different species collected in Pakistan and in China, including wild species of Ziziphus mauritiana, Z. nummularia and Z. oxyphllya, and both wild and domesticated Z. jujuba Mill. Population structure, phylogenetic analysis and principal coordinates analysis based on 10,889 high-quality SNPs derived from genotyping-by-sequencing indicated that the accessions clustered into two major groups. The wild Pakistani Z. jujuba and Z. nummularia exhibited higher genetic diversity and polymorphic information content (0.37 and 0.23 respectively) than other species. We further conducted a genome-wide association study and discovered six highly significant marker-trait associations for fruit weight and plant height in this population. Our study provides important information for future breeding of Ziziphus species.


title: Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

abstract:
PurposeTargeted regulation of transfected extra-chromosomal plasmid DNA typically requires the integration of 9 - 20 bp docking sites into the plasmid. Here, we report an elegant approach, The Dpn Adaptor Linked Effector (DAL-E) system, to target fusion proteins to 6-methyladenosine in GATC, which appears frequently in popular eukaryotic expression vectors and is absent from endogenous genomic DNA. Methods: The DNA-binding region from the DpnI endonuclease binds 6-methyladenosine within the GATC motif. We used a Dpn-transcriptional activator (DPN7-TA) fusion to induce gene expression from transiently transfected pDNAs.

ResultsWe validated methylation-dependent activity of DPN7-TA with a panel of target pDNAs. We observed stronger transactivation when GATC targets were located upstream of the transcriptional start site in the target pDNA. Conclusion: DAL-E, consisting of a 108 aa, 12 kD DNA-binding adaptor and a 4 bp recognition site, offers a genetically-tractable, tunable system that can potentially be redesigned to recruit a variety of regulators (e.g. activators, silencers, epigenome editors) to transfected plasmid DNA.

LAY SUMMARYTransfection of plasmid DNA (pDNA) is a commonly used method for introducing exogenous genetic material into mammalian cells. Once introduced into cells not all pDNAs express this genetic material at sufficient levels. Current techniques to improve transgene expression are limited and are not always feasible for all plasmids. This report presents a new method to improve gene expression from pDNA. The Dpn Adaptor Linked Effector (DAL-E) binds to methylated adenines in the pDNA resulting in increased expression. This technique has exciting implications for improved genetic engineering of mammalian cells.

GRAPHICAL ABSTRACT

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category: synthetic biology


title: Optimized immunoglobulin knock-ins using Cas9 reveal peritoneal B cell lineage relationships in vivo

abstract: Immunoglobulin (Ig) knock-in mice are valuable tools in basic and translational immunological research. Here we present "Speed-Ig," a rapid Cas9-based method for generating Ig knock-in mouse lines with high on-target integration rates at both heavy and light chain alleles. With standardized target sites and promoter regions, Speed-Ig mice can be used for comparative studies of B cell biology and vaccine optimization in vivo. We used Speed-Ig to create panels of mice with Ig pairs derived from B-1a, B-1b, and B-2 cells. Surprisingly, B-1b and B-2 Ig pairs drove both B-1b and B-2 phenotypes, suggesting a previously unknown lineage relationship between these subsets. We then confirmed the B-1:B-2 relationship with transcription factor reporter lines and through adoptive cell transfer experiments. In summary, our Ig knock-in approach facilitated the discovery of previously unappreciated aspect of innate-like B cell biology.


title: SARS-CoV-2 mechanistic correlates of protection: insight from modelling response to vaccines

abstract:
The definition of correlates of protection is critical for the development of next generation SARS-CoV-2 vaccine platforms. Here, we propose a new framework for identifying mechanistic correlates of protection based on mathematical modelling of viral dynamics and data mining of immunological markers. The application to three different studies in non-human primates evaluating SARS-CoV-2 vaccines based on CD40-targeting, two-component spike nanoparticle and mRNA 1273 identifies and quantifies two main mechanisms that are a decrease of rate of cell infection and an increase in clearance of infected cells. Inhibition of RBD binding to ACE2 appears to be a robust mechanistic correlate of protection across the three vaccine platforms although not capturing the whole biological vaccine effect. The model shows that RBD/ACE2 binding inhibition represents a strong mechanism of protection which required significant reduction in blocking potency to effectively compromise the control of viral replication.

One Sentence SummaryA framework for modelling the immune control of viral dynamics is applied to quantify the effect of several SARS-CoV-2 vaccine platforms and to define mechanistic correlates of protection.


title: Increased RNA Polymerase Activity and Pausing at CTCF binding sites on the Epstein Barr Virus Genome During Reactivation from Latency

abstract:
The ability of Epstein-Barr Virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent to lytic switch we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in latently infected Mutu-I cells at 1, 4 and 12 h post-reactivation. During latency, Pol activity was primarily limited to the EBNA1 transcript initiating at the Qp promoter, the EBER and RPMS1/BART regions and the BHLF1 transcript. Unexpectedly at early time-points post-reactivation, the EBV transcripts with the largest increase in Pol activity were LMP-2A, EBER-1 and RPMS1. Closer analysis of the PRO-Seq data at these regions revealed a distinct pattern of high Pol activity with bidirectional transcription and strong peaks indicative of Pol pausing. Alignment to ChIP-Seq data revealed a strong correlation with CTCF binding sites on the EBV genome. In addition, alignment to ATAC-Seq data indicated that many of these transcription regulatory regions were sites of accessible chromatin. Similar features were observed in Akata cells activated from latency with anti-IgG. Overall, these data suggest that during reactivation, EBV recruits RNA polymerase to CTCF binding sites where it transcribes short distances and pauses. These activities likely help open chromatin on the viral genome to initiate productive replication.

Author summaryThe ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B-cells and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remains unknown. In this study, we were able to precisely map RNA polymerase positioning and activity during latency and reactivation. Unexpectedly, RNA polymerase activity was not enriched at the IE genes during early reactivation but accumulated at distinct regions, characteristic of enhancers, on the EBV genome previously shown to be associated with CTCF and open chromatin. We speculate that Pol accumulation at these sites helps maintain open chromatin locally, eventually leading to changes in the quaternary structure of the EBV genome to promote lytic replication.


title: Black-necked spitting cobra (Naja nigricollis) phospholipases A2 cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket

abstract: African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to a glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components including proteases and phospholipases. Here, we investigated the effect of Naja nigricollis on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VGS hydrolysis however N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) a cell cycle arrest prior to cytokinesis. A single protein family, the phospholipases A2s present in N. nigricollis venom, was necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.


title: Bacterial growth in multicellular aggregates leads to the emergence of complex lifecycles

abstract: In response to environmental stresses such as starvation, many bacteria facultatively aggregate into multicellular structures that can attain new metabolic functions and behaviors. Despite the ubiquity and relevance of this form of collective behavior, we lack an understanding of how the spatiotemporal dynamics of aggregate development emerge from cellular physiology. Here, we show that the coupling between growth and spatial gradient formation leads to the emergence of a complex lifecycle, akin to those known for multicellular bacteria. Under otherwise carbon-limited growth conditions, the marine bacterium Vibrio splendidus 12B01 forms multicellular groups to collectively harvest carbon from the brown-algal polysaccharide alginate. This is achieved during growth on dissolved alginate polymer through formation of spherical, clonal clusters of cells that grow up to 40 {micro}m in radius. Clusters develop striking spatial patterning as they grow due to phenotypic differentiation of sub-populations into a  shell of static cells surrounding a motile  core. Combining in situ measurements of cell physiology with transcriptomics, we show that shell cells express adhesive type IV pili, while motile core cells express carbon storage granules. The emergence of shell and core phenotypes is cued by opposing gradients of carbon and nitrogen that form within cell clusters due to local metabolic activity. Eventually, the shell ruptures, releasing the carbon-storing core, and we show that carbon-storing cells more readily propagate on alginate than non-carbon storing cells. We propose that phenotypic differentiation promotes the resilience of 12B01 groups by enabling clonal groups to grow larger and propagate more effectively. Phenotypic differentiation may be a widespread, but overlooked, strategy among bacteria to enhance resilience in the context of resource limitation.


title: Antibiotic inhibition of the Plasmodium apicoplast decreases haemoglobin degradation and antagonises dihydoartemisinin action

abstract: The World Health Organisation (WHO) recommends artemisinin (ART) combinations for treatment of uncomplicated Plasmodium falciparum malaria. Understanding the interaction between co-administered drugs within combination therapies is clinically important to prevent unintended consequences. The WHO guidelines recommend second line treatments that combine artesunate with tetracycline, doxycycline, or clindamycin--antibiotics that target the Plasmodium relict plastid, the apicoplast. In addition, antibiotics can be used simultaneously against other infectious diseases, leading to their inadvertent combination with ARTs. One consequence of apicoplast inhibition is a perturbation to haemoglobin uptake and trafficking--a pathway required for activation of ART derivatives. Here, we show that apicoplast-targeting antibiotics reduce the abundance of the catalyst of ART activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between ART and these antibiotics, suggesting that apicoplast inhibitors reduce ART activation. These data have potential clinical implications due to the reliance on--and widespread use of--both ARTs and these antibiotics in malaria endemic regions.


title: Antibiotic inhibition of the Plasmodium apicoplast decreases haemoglobin degradation and antagonises dihydroartemisinin action


title: An essential ER-resident N-acetyltransferase in Plasmodium falciparum

abstract: N-terminal acetylation is a common eukaryotic protein modification that involves the addition of an acetyl group to the N-terminus of a polypeptide. This modification is largely performed by cytosolic N-terminal acetyltransferases (NATs). Most associate with the ribosome, acetylating nascent polypeptides co-translationally. In the malaria parasite Plasmodium falciparum, exported effectors are translated into the ER, processed by the aspartic protease plasmepsin V and then N-acetylated, despite having no clear access to cytosolic NATs. Here, we used post-transcriptional knockdown to investigate the most obvious candidate, Pf3D7_1437000. We found that it co-localizes with the ER-resident plasmepsin V and is required for parasite growth. However, depletion of Pf3D7_1437000 had no effect on protein export or acetylation of the exported proteins HRP2 and HRP3. Pf3D7_1437000-depleted parasites arrested later in their development cycle than export-blocked parasites, suggesting the proteins essential role is distinct from protein export.


title: A universal, single primer amplification protocol (R-SPA) to perform whole genome sequencing of segmented dsRNA reoviruses

abstract:
BackgroundThe Reoviridae family represents the largest family of double-stranded RNA (dsRNA) viruses, and the members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals, and birds. Genomic characterization of reoviruses has not been adopted on a large-scale due to the complexity of obtaining sequences for all 10 segments.

ResultsIn this study, we developed a time-efficient, and practical method to enrich reovirus sequencing reads from isolates that allowed for full genome recovery using single-primer amplification method coupled with next generation sequencing. We refer to this protocol as reovirus-Single Primer Amplification (R-SPA). Our results demonstrated that most of the genes were covered with at least 500 reads per base space. Furthermore, R-SPA covered both 5 and 3 end of each reovirus genes.

ConclusionA universal and fast amplification protocol that yields double-stranded cDNA in sufficient abundance and facilitates and expedites the whole genome sequencing of reoviruses was presented in this study.


title: A new gene family diagnostic for intracellular biomineralization of amorphous Ca-carbonates by cyanobacteria

abstract:
Cyanobacteria have massively contributed to carbonate deposit formation over the geological history. They are traditionally thought to biomineralize CaCO3 extracellularly as an indirect byproduct of photosynthesis. However, the recent discovery of freshwater cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges this view. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and evolutionary history remain elusive. Here, using comparative genomics, we identify a new gene (ccyA) and protein (calcyanin) family specifically associated with cyanobacterial iACC biomineralization. Calcyanin is composed of a conserved C-terminal domain, which likely adopts an original fold, and a variable N-terminal domain whose structure allows differentiating 4 major types among the 35 known calcyanin homologues. Calcyanin lacks detectable full-length homologs with known function. Yet, genetic and comparative genomic analyses suggest a possible involvement in Ca homeostasis, making this gene family a particularly interesting target for future functional studies. Whatever its function, this new gene family appears as a gene diagnostic of intracellular calcification in cyanobacteria. By searching for ccyA in publicly available genomes, we identified 13 additional cyanobacterial strains forming iACC. This significantly extends our knowledge about the phylogenetic and environmental distribution of cyanobacterial iACC biomineralization, especially with the detection of multicellular genera as well as a marine species. Phylogenetic analyses indicate that iACC biomineralization is ancient, with independent losses in various lineages and some HGT cases that resulted in the broad but patchy distribution of calcyanin across modern cyanobacteria. Overall, iACC biomineralization emerges as a new case of genetically controlled biomineralization in bacteria.

Significance statementFew freshwater species of Cyanobacteria have been known to mineralize amorphous CaCO3 (ACC) intracellularly. Despite the geochemical interest of this biomineralization, its evolutionary history and molecular mechanism remain poorly known. Here, we report the discovery of a new gene family that has no homolog with known function, which proves to be a good diagnostic marker of this process. It allowed to find cyanobacteria in several phyla and environments such as seawater, where ACC biomineralization had not been reported before. Moreover, this gene is ancient and was independently lost in various lineages with some later horizontal transfers, resulting in a broad and patchy phylogenetic distribution in modern cyanobacteria.

category: evolutionary biology


title: Nuclear activation in dual-durotaxing cells on a matrix with cell-scale stiffness-heterogeneity

abstract: Living organisms are typically composed of various tissues with microscopic cell-scale stiffness-heterogeneity, in which some cells receive dynamically fluctuating mechanical stimuli from the heterogeneous extracellular milieu during long-term movement. Although intracellular stress dynamics (ISD), which are closely related to the regulation of cell functions such as proliferation and differentiation, can be characteristically modulated in cells migrating on a matrix with stiffness-heterogeneity, it has been unclear how the mode of fluctuation of ISD affects cell functions. In the present study, we demonstrate that mesenchymal stem cells (MSCs) dual-durotaxing (i.e., both forward and reverse durotaxis) on microelastically-patterned gels with stiff triangular domains markedly amplify the fluctuation of ISD, nuclear shape, and the spatial distribution of chromatins, which makes the cells remain far from tensional equilibrium. We provide evidence that amplified chromatin fluctuation in the dual-durotaxing MSCs can cause activation of cellular vigor and maintenance of the stemness.


title: Intratumoral delivery of engineered recombinant modified vaccinia virus Ankara express-ing Flt3L and OX40L generates potent antitumor immunity through activating the cGAS/STING pathway and depleting tumor-infiltrating regulatory T cells

abstract:
Intratumoral (IT) delivery of immune-activating viruses can serve as an important strategy to turn "cold" tumors into "hot" tumors, resulting in overcoming resistance to immune checkpoint blockade (ICB). Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus that has a long history of human use. Here we report that IT recombinant MVA (rMVA), lacking E5R encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase (cGAS), expressing a dendritic cell growth factor, Fms-like tyrosine kinase 3 ligand (Flt3L), and a T cell co-stimulator, OX40L, generates strong antitumor immunity, which is dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and STAT1/STAT2-mediated type I IFN signaling. Remarkably, IT rMVA depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Taken together, our study provides a proof-of-concept for improving MVA-based cancer immunotherapy, through modulation of both innate and adaptive immunity.

One Sentence SummaryIntratumoral delivery of recombinant MVA for cancer immunotherapy


title: A metagenomic analysis of the wrackbed microbiome indicates a phylogeographic break along the North Sea - Baltic Sea transition zone

abstract: Sandy beaches are biogeochemical hotspots that bridge marine and terrestrial ecosystems via the transfer of marine organic matter, such as seaweed (termed wrack). A keystone of this unique ecosystem is the microbial community, which helps to degrade wrack and re-mineralize nutrients. However, little is known about the wrackbed microbiome, its composition, trophic ecology, or how it varies over time and space. Here we characterize the wrackbed microbiome as well as the microbiome of a primary consumer, the seaweed fly Coelopa frigida, and examine how they change along one of the most studied ecological gradients in the world, the transition from the marine North Sea to the brackish Baltic Sea. We found that polysaccharide degraders dominated both the wrackbed and seaweed fly microbiomes but there were still consistent differences between wrackbed and fly samples. Furthermore, we observed a shift in both microbial communities and functionality between the North and Baltic Sea. These shifts were mostly due to changes in the frequency of different groups of known polysaccharide degraders (Proteobacteria and Bacteroidota). We hypothesize that microbes were selected for their abilities to degrade different polysaccharides corresponding to a shift in polysaccharide content in the seaweed communities of the North vs. Baltic Sea. Our results reveal the complexities of both the wracked microbial community, with different groups specialized to different roles, and the cascading trophic consequences of shifts in the near shore algal community.


title: Microbiome restructuring: dominant coral bacterium Endozoicomonas species display differential adaptive capabilities to environmental changes

abstract: Bacteria in the coral microbiome play a crucial role in determining coral health and fitness, and the coral host often restructures its microbiome composition in response to external factors. An important but often neglected factor determining this microbiome restructuring is the capacity of microbiome members to adapt to a new environment. To address this issue, we examined how the microbiome structure of Acropora muricata corals changed over 9 months following a reciprocal transplant experiment. Using a combination of metabarcoding, genomics, and comparative genomics approaches, we found that coral colonies separated by a small distance harbored different dominant Endozoicomonas related phylotypes belonging to two different species, including a novel species, Candidatus Endozoicomonas penghunesis 4G, whose chromosome level (complete) genome was also sequenced in this study. Furthermore, the two dominant Endozoicomonas species showed varied adaptation capabilities when coral colonies were transplanted in a new environment. The differential adaptation capabilities of dominant members of the microbiome can a) provide distinct advantages to coral hosts when subjected to changing environmental conditions and b) have positive implications for future reefs.


title: Flexible circuit mechanisms for context-dependent song sequencing

abstract: Many sequenced behaviors, including locomotion, reaching, and vocalization, are patterned differently in different contexts, enabling animals to adjust to their current environments. However, how contextual information shapes neural activity to flexibly alter the patterning of actions is not yet understood. Prior work indicates such flexibility could be achieved via parallel motor circuits, with differing sensitivities to sensory context [1, 2, 3]; instead we demonstrate here how a single neural pathway operates in two different regimes dependent on recent sensory history. We leverage the Drosophila song production system [4] to investigate the neural mechanisms that support male song sequence generation in two contexts: near versus far from the female. While previous studies identified several song production neurons[5, 6, 7, 8], how these neurons are organized to mediate song patterning was unknown. We find that male flies sing  simple trains of only one syllable or mode far from the female but complex song sequences consisting of alternations between modes when near to her. We characterize the male song circuit from the brain to the ventral nerve cord (VNC), and find that the VNC song pre-motor circuit is shaped by two key computations: mutual inhibition and rebound excitability [9] between nodes driving the two modes of song. Weak sensory input to a direct brain-to-VNC excitatory pathway (via pC2 brain and pIP10 descending neurons) drives simple song far from the female. Strong sensory input to the same pathway enables complex song production via simultaneous recruitment of P1a neuron-mediated disinhibition of the VNC song pre-motor circuit. Thus, proximity to the female effectively unlocks motor circuit dynamics in the correct sensory context. We construct a compact circuit model to demonstrate that these few computations are sufficient to replicate natural context-dependent song dynamics. These results have broad implications for neural population-level models of context-dependent behavior [10] and highlight that canonical circuit motifs [11, 12, 13] can be combined in novel ways to enable circuit flexibility required for dynamic communication.


title: Coherent theta oscillations in the cerebellum and supplementary motor area mediate visuomotor adaptation

abstract: The cerebellum and its interaction with cortical areas play a key role in our ability to flexibly adapt a motor program in response to sensory input. Current knowledge about specific neural mechanisms underlying the process of visuomotor adaptation is however lacking. Using a novel placement of EEG electrodes to record electric activity from the cerebellum, we studied local cerebellar activity, as well as its coupling with neocortical activity to obtain direct neurophysiological markers of visuomotor adaptation in humans. We found increased theta (4-8Hz) power in "cerebellar" as well as cortical electrodes, when subjects first encountered a visual perturbation. Theta power decreased as subjects adapted to the perturbation, and rebounded when the perturbation was suddenly removed. This effect was observed in two distinct locations: a cerebellar cluster and a central cluster, which were localized in left cerebellar crus I (lCB) and right supplementary motor area (rSMA) using linear constrained minimum variance beamforming. Importantly, we found that better adaptation was associated with increased theta power in left cerebellar electrodes and a right sensorimotor cortex electrode. Finally, increased rSMA -> lCB connectivity was significantly decreased with adaptation. These results demonstrate that: (1) cerebellar theta power is markedly modulated over the course of visuomotor adaptation and (2) theta oscillations could serve as a key mechanism for communication within a cortico-cerebellar loop.


title: Multilevel neural gradients reflect transdiagnostic effects of major psychiatric conditions on cortical morphology

abstract: AO_SCPLOWBSTRACTC_SCPLOWIt is increasingly recognized that multiple psychiatric conditions are underpinned by shared neural pathways, affecting similar brain systems. Here, we assessed i) shared dimensions of alterations in cortical morphology across six major psychiatric conditions (autism spectrum disorder, attention deficit/hyperactivity disorder, major depression, obsessive-compulsive disorder, bipolar disorder, schizophrenia) and ii) carried out a multiscale neural contextualization, by cross-referencing shared anomalies against cortical myeloarchitecture and cytoarchitecture, as well as connectome and neurotransmitter organization. Pooling disease-related effects on MRI-based cortical thickness measures across six ENIGMA working groups, including a total of 28,546 participants (12,876 patients and 15,670 controls), we computed a shared disease dimension on cortical morphology using principal component analysis that described a sensory-fugal pattern with paralimbic regions showing the most consistent abnormalities across conditions. The shared disease dimension was closely related to cortical gradients of microstructure and intrinsic connectivity, as well as neurotransmitter systems, specifically serotonin and dopamine. Our findings embed the shared effects of major psychiatric conditions on brain structure in multiple scales of brain organization and may provide novel insights into neural mechanisms into transdiagnostic vulnerability.


title: Inhibition of SAR S-CoV-2 infection and replication by lactoferrin, MUC1 and α-lactalbumin identified in human breastmilk

abstract: The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is an extremely complex with nutritional composition to nourish infants and protect them from different kinds of infection diseases and also SARS-CoV-2 infection. Previous studies have found that breastmilk exhibited potent antiviral activity against SARS-CoV-2 infection. However, it is still unknown which component(s) in the breastmilk is responsible for its antiviral activity. Here, we identified Lactoferrin (LF), MUC1 and -Lactalbumin (-LA) from human breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and in vitro confirmation that inhibited SARS-CoV-2 infection and analyzed their antiviral activity using the SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) in the Huh7.5, Vero E6 and Caco-2-N cell lines. Additionally, we found that LF and MUC1 could inhibit viral attachment, entry and post-entry replication, while -LA just inhibit viral attachment and entry. Importantly, LF, MUC1 and -LA possess potent antiviral activities towards not only wild-type but also variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Moreover, LF from other species (e.g., bovine and goat) is still capable of blocking viral attachment to cellular heparan sulfate. Taken together, our study provided the first line of evidence that human breastmilk components (LF, MUC1 and -LA) are promising therapeutic candidates warranting further development or treatingVID-19 given their exceedingly safety levels.


title: A database and comprehensive analysis of the algae genomes

abstract: Algae characterize their high diversity, taxonomy and morphology for wide-used studying the plant origins and terrestrialization, as well as multicellular evolution. Due to the genome assembly challenge of algae caused by symbionts with microbiome, the published algae genomes are relatively less than the terrestrial plants. Here we comprehensively collected and re-annotated 191 available algae genomes distributed in nine major lineages. We systemically investigated the genome features including genome size, assembly continuity and integrity, GC content, abundance of repetitive sequences and protein-coding gene number. We construct the phylogenetic trees using 193 algae genomes, which is consistent with the well-known evolution path that Glaucophyte is the most ancient, going through eight lineages, and finally evolved to terrestrial plants. We also examined the Horizontal Gene Transfer (HGT) genes distribution in algae genomes and provides a substantial genomic resource for functional gene origins and plant evolution.


title: The relationship between human thymus-derived regulatory T cells, TGF-β-induced regulatory T cells and conventional CD4+ T cells as revealed by proteomics

abstract: The CD4+ regulatory T (Treg) cell lineage, as defined by FOXP3 expression, comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. In human, naive tTreg cells can be isolated from blood, while effector tTreg and pTreg cells cannot be purified reliably for lack of cell surface markers. As a model for Treg cells, studies often employ TGF-{beta}-induced (i)Treg cells generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells, as optimally purified from human blood. Proteomic analysis revealed that each of these cell populations has a unique protein expression pattern before and after CD3/CD28-mediated activation. iTreg cells had limited overlap in protein expression with tTreg cells and exhibited markedly differential expression of proteins involved in signal transduction and metabolism. Whereas iTreg and Tconv cells responded strongly to CD3/CD28-mediated activation, tTreg cells showed a modest response, which reflects their adaptations in signal transduction pathways. As a benchmark, we used a previously defined proteomic signature that discerns ex vivo naive and effector Treg cells from Tconv cells and includes conserved Treg cell properties. This Treg cell core signature was largely absent in iTreg cells, as exemplified by STAT4 expression that may contribute to pro-inflammatory functions. In addition, we used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naive Treg cells. This effector Treg cell signature was partially present in iTreg cells, indicating that they do have protein expression features in common with ex vivo effector Treg cells. In conclusion, iTreg cells are distinct from tTreg cells and largely lack the Treg cell core proteomic signature, indicating that caution is warranted when using iTreg cells as a model for Treg cell populations found in vivo.


title: Proteomics reveals unique identities of human TGF-β-induced and thymus-derived CD4+ regulatory T cells


title: Interpretable Visualization of Scientific Hypotheses in Literature-based Discovery

abstract: In this paper we present an approach for interpretable visualization of scientific hypotheses that is based on the idea of semantic concept interconnectivity, network-based and topic modeling methods. Our visualization approach has numerous adjustable parameters which provides the domain experts with additional flexibility in their decision making process. We also make use of the Unified Medical Language System metadata by integrating it directly into the resulting topics, and adding the variability into hypotheses resolution. To demonstrate the proposed approach in action, we deployed end-to-end hypothesis generation pipeline AGATHA, which was evaluated by BioCreative VII experts with COVID-19-related queries.

category: bioinformatics


title: Diverse operant control of different motor cortex populations during learning

abstract: During motor learning, as well as during neuroprosthetic learning, animals learn to control motor cortex activity in order to generate behavior. Two different population of motor cortex neurons, intra-telencephalic (IT) and pyramidal tract (PT) neurons, convey the resulting cortical signals within and outside the telencephalon. Although a large amount of evidence demonstrates contrasting functional organization among both populations, it is unclear whether the brain can equally learn to control the activity of either class of motor cortex neurons. To answer this question, we used a Calcium imaging based brain-machine interface (CaBMI) and trained different groups of mice to modulate the activity of either IT or PT neurons in order to receive a reward. We found that animals learn to control PT neuron activity faster and better than IT neuron activity. Moreover, our findings show that the advantage of PT neurons is the result of characteristics inherent to this population as well as their local circuitry and cortical depth location. Taken together, our results suggest that motor cortex is optimized to control the activity of pyramidal track neurons, embedded deep in cortex, and relaying motor commands outside of the telencephalon.


title: Two parallel pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans

abstract: Ultrasound has been shown to affect the function of both neurons and non-neuronal cells. However, the underlying molecular machinery has been poorly understood. Here, we show that at least two mechanosensitive proteins act in parallel to generate C. elegans behavioral responses to ultrasound stimuli. We first show that these animals generate reversals in response to a single 10 msec pulse from a 2.25 MHz ultrasound transducer. Next, we show that the pore-forming subunit of the mechanosensitive channel TRP-4, and a DEG/ENaC/ASIC ion channel MEC-4, are both required for this ultrasound-evoked reversal response. Further, the trp-4 mec-4 double mutant shows a stronger behavioral deficit compared to either single mutant. Finally, overexpressing TRP-4 in specific chemosensory neurons can rescue the ultrasound-triggered behavioral deficit in the mec-4 null mutant, suggesting that these two pathways act in parallel. Together, we demonstrate that multiple mechanosensitive proteins likely cooperate to transform ultrasound stimuli into behavioral changes.


title: Bimodal Remapping of Visual Grids

abstract:
Spatially modulated neurons from the rat secondary visual cortex (V2) show grid-like firing patterns during freely foraging in open-field enclosures. However, the remapping of the V2 grid cells is not well understood. Here we report two classes of V2 grid cell populations with distinct remapping properties: one regular class with invariant grid field patterns, and the other bimodal class that has remapping induced by environmental manipulations such as changes in enclosure shape, size, orientation and lighting in a familiar environment. The bimodal V2 grid cell pattern remains stable regardless of the follow-up manipulations, but restores to the original firing pattern upon animals re-entry into the familiar environment on the next day or from the novel environment. The bimodal V2 grid cells are modulated with theta frequency during the course of remapping and stabilize quickly. We also found conjunctive bistable V2 grid cells with invariant head directional tuning. Overall, our results suggest a new grid cell mechanism in V2 that is different from the medial entorhinal cortex (MEC) grid cells.

HighlightsO_LIBistable V2 grid cells display bimodal or bistable remapping
C_LIO_LIV2 grid cell firing patterns are not disrupted in darkness
C_LIO_LIV2 grid cells preserve theta frequency modulation during remapping
C_LI


title: Measuring optimality of a neural system by a logic gate model

abstract: The nematode worm Caenorhabditis elegans has a relatively simple neural system for analysis of information transmission from sensory organ to muscle fiber. Therefore, an example of a neural circuit is analyzed that originates in the nematode worm, and a method is applied for measuring its information flow efficiency by use of a model of logic gates. This model-based approach is useful where the assumptions of a logic gate design are applicable. It is also an useful approach where there are competing mathematical models for explaining the role of a neural circuit since the logic gate model can estimate the computational complexity of a network, and distinguish which of the mathematical models require fewer computations. In addition, for generalization of the concept of information optimality in biological systems, there is an extensive discussion of its role in the genetic-based pathways of organisms.


title: A perspective on information optimality in a neural circuit


title: MutationalPatterns: The one stop shop for the analysis of mutational processes

abstract:
BackgroundThe collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data.

ResultsHere, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights.

ConclusionsThis novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns.


title: Dietary adaptation in Neandertal, Denisovan and Sapiens revealed by gene copy number variation

abstract: Dietary adaptation is the acquisition of an efficient system to digest food available in an ecosystem. To find the genetic basis for human dietary adaptation, we searched 16 genomes from Neandertal, Denisovan and Early Sapiens for food digestion genes that tend to have more or fewer copies than the modern human reference genome. Here, we identify 11 genes, including three gene clusters, with discernible copy number variation trends at the population level. The genomic variation shows how metabolic pathways for lipid, brown fat, protein or carbohydrate metabolism adapt to metabolize food from animal or plant sources. Interpreting the copy number profiles in relation to fossil evidence shows that Homo sapiens had an evolutionary advantage compared to Neandertal and Denisovan in adapting to cold and temperate ecosystems.


title: Diversity, function and evolution of aquatic vertebrate genomes

abstract: Aquatic vertebrates consist of jawed fish (cartilaginous fish and bony fish), aquatic mammals, reptiles and amphibians. Here, we present a comprehensive analysis of 630 aquatic vertebrate genomes to generate a standardized compendium of genomic data. We demonstrate its value by assessing their genome features as well as illuminating gene families related to the transition from water to land, such as Hox genes and olfactory receptor genes. We found that LINEs are the major transposable element (TE) type in cartilaginous fish and aquatic mammals, while DNA transposons are the dominate type in bony fish. To our surprise, TE types are not fixed in amphibians, the first group that transitioned to living on land. These results illustrate the value of a unified resource for comparative genomic analyses of aquatic vertebrates. Our data and strategy are likely to support all evolutionary and ecological research on vertebrates.