title: Survey on Open Science Practices in Functional Neuroimaging

abstract:
Replicability and reproducibility of scientific findings is paramount for sustainable progress in neuroscience. Preregistration of the hypotheses and methods of an empirical study before analysis, the sharing of primary research data, and compliance with data standards such as the Brain Imaging Data Structure (BIDS), are considered effective practices to secure progress and to substantiate quality of research. We investigated the current level of adoption of open science practices in neuroimaging and the difficulties that prevent researchers from using them.

Email invitations to participate in the survey were sent to addresses received through a PubMed search of human functional magnetic resonance imaging studies between 2010 and 2020. 283 persons completed the questionnaire.

Although half of the participants were experienced with preregistration, the willingness to preregister studies in the future was modest. The majority of participants had experience with the sharing of primary neuroimaging data. Most of the participants were interested in implementing a standardized data structure such as BIDS in their labs. Based on demographic variables, we compared participants on seven subscales, which had been generated through factor analysis. It was found that experienced researchers at lower career level had higher fear of being transparent, researchers with residence in the EU had a higher need for data governance, and researchers at medical faculties as compared to other university faculties reported a higher need for data governance and a more unsupportive environment.

The results suggest growing adoption of open science practices but also highlight a number of important impediments.

category: scientific communication and education


title: Neural entrainment is strongest to the spectral flux of slow music and depends on familiarity and beat salience

abstract: Neural activity in the auditory system synchronizes to sound rhythms, and brain-environment synchronization is thought to be fundamental to successful auditory perception. Sound rhythms are often operationalized in terms of the sounds amplitude envelope. We hypothesized that - especially for music - the envelope might not best capture the complex spectro-temporal fluctuations that give rise to beat perception and synchronize neural activity. This study investigated 1) neural entrainment to different musical features, 2) tempo-dependence of neural entrainment, and 3) dependence of entrainment on familiarity, enjoyment, and ease of beat perception. In this electroencephalography study, 37 human participants listened to tempo-modulated music (1-4 Hz). Independent of whether the analysis approach was based on temporal response functions (TRFs) or reliable components analysis (RCA), the spectral flux of music - as opposed to the amplitude envelope - evoked strongest neural entrainment. Moreover, music with slower beat rates, high familiarity, and easy-to-perceive beats elicited the strongest neural response. Based on the TRFs, we could decode music stimulation tempo, but also perceived beat rate, even when the two differed. Our results demonstrate the importance of accurately characterizing musical acoustics in the context of studying neural entrainment, and demonstrate entrainments sensitivity to musical tempo, familiarity, and beat salience.

category: neuroscience


title: How synchrony and metastable network dynamics are affected in fast and slow timescales with aging: Implication for Cognition

abstract: Both healthy and pathological aging exhibits gradual deterioration of structure but in-terestingly in healthy aging adults often maintains a high level of cognitive performance in a variety of cognitively demanding task till late age. What are the relevant network measures that could possibly track these dynamic changes which may be critically relevant for maintenance of cognitive functions through lifespan and how does these measures affected by the specific alterations in underlying anatomical connectivity till day remains an open question. In this work, we propose that whole-brain computational models are required to test the hypothesis that aging affects the brain network dynamics through two highly relevant network measures synchrony and metastability. Since aging entails complex processes involving multiple timescales we test the additional hypothesis that whether these two network measures remain invariant or exhibit different behavior in the fast and slow timescales respectively. The altered global synchrony and metastability with aging can be related to shifts in the dynamic working point of the system based on biophysical parameters e.g., time delay, and inter-areal coupling constrained by the underlying structural connectivity matrix.Using diffusion tensor imaging (DTI) data, we estimate structural connectivity (SC) of individual group of participants and obtain network level synchrony, metastability indexing network dynamics from resting state functional MRI data for both young and elderly participants in the age range of 18-89 years. Subsequently, we simulate a whole-brain Kuramoto model of coupled oscillators with appropriate conduction delay and interareal coupling strength to test the hypothesis of shifting of dynamic working point with age-associated alteration in network dynamics in both neural and ultraslow BOLD signal time scales. Specifically, we investigate the age-associated difference in metastable brain dynamics across large-scale neurocognitive brain networks e.g., salience network (SN), default mode network (DMN), and central executive network (CEN) to test spatio-temporal changes in default to executive coupling hypothesis with age. Interestingly, we find that the metastability of the SN increases substantially with age, whereas the metastability of the CEN and DMN networks do not substantially vary with age suggesting a clear role of conduction delay and global coupling in mediating altered dynamics in these networks. Moreover, our finding suggests that the metastability changes from slow to fast timescales confirming previous findings that variability of brain signals relates differently in slower and faster time scales with aging. However, synchrony remains invariant network measure across timescales and agnostic to the filtering of fast signals. Finally, we demonstrate both numerically and analytically longrange anatomical connections as oppose to shot-range or mid-range connection alterations is responsible for the overall neural difference in large-scale brain network dynamics captured by the network measure metastability. In summary, we propose a theoretical framework providing a systematic account of tracking age-associated variability and synchrony at multiple time scales across lifespan which may pave the way for developing dynamical theories of cognitive aging.


title: Adolescent ethanol drinking promotes hyperalgesia, neuroinflammation and serotonergic deficits in mice that persist into adulthood

abstract: Adolescent alcohol use can permanently alter brain function and lead to poor health outcomes in adulthood. Emerging evidence suggests that alcohol use predispose to pain disorders or exacerbate existing pain conditions, but the neural mechanisms are currently unknown. Here we report that mice exposed to adolescent intermittent access to ethanol (AIE) exhibit increased pain sensitivity and depressive-like behaviors that persist after alcohol cessation and are accompanied by elevated CD68 expression in microglia and reduced numbers of serotonin (5-HT)-expressing neurons in the dorsal raphe nucleus (DRN). 5-HT expression was also reduced in the thalamus, anterior cingulate cortex (ACC) and amygdala as well as the lumbar dorsal horn of the spinal cord. We then found that chronic minocycline administration after AIE alleviated hyperalgesia and social deficits, while chemogenetic activation of microglia in the DRN of Cx3cr1-cre-GFP mice reproduced the effects of AIE on pain and social interaction. Taken together, these results indicate that microglial activation in the DRN may be a primary driver of pain and negative affect after AIE.


title: Density-by-diet interactions during larval development shape adult life-history trait expression and fitness in a polyphagous fly

abstract: Habitat quality early in life determines individual fitness, with possible long-term evolutionary effects on groups and populations. In holometabolous insects, larval ecology plays a major role in determining the expression of traits in adulthood, but how ecological conditions during larval stage interact to shape adult life-history and fitness, particularly in non-model organisms, remains subject to scrutiny. Consequently, our knowledge of the interactive effects of ecological factors on insect development is limited. Here, using the polyphagous fly Bactrocera tryoni, we conducted a fully-factorial design where we manipulated larval density and larval diet (protein-rich, standard, and sugar-rich) to gain insights into how these ecological factors interact to modulate adult fitness. As expected, a protein-rich diet resulted in faster larval development, heavier and leaner adults that were more fecund compared with standard and sugar-rich diets, irrespective of larval density. Females from the protein-rich larval diet had overall higher reproductive rate (i.e., eggs per day) than females from other diets, and reproductive rate decreased linearly with density for females from the protein-rich but non-linearly for females from the standard and sugar-rich diets over time. Surprisingly, adult lipid reserve increased with larval density for adults from the sugar-rich diet (as opposed to decreasing, as in other diets), possibly due to a stress-response to an extremely adverse condition during development (i.e., high intraspecific competition and poor nutrition). Together, our results provide insights into how ecological factors early in life interact and shape the fate of individuals through life-stages in holometabolous insects.

category: zoology


title: Protective immunity of the primary SARS-CoV-2 infection reduces disease severity post re-infection with Delta variants in Syrian hamsters

abstract:
Delta variant has evolved to become dominant SARS-CoV-2 lineage worldwide and there are reports of secondary infections with varying severity in vaccinated and unvaccinated naturally recovered COVID-19 patients. As the protective immunity following the infection wanes within few months, studies of re-infection after prolonged duration is needed. Hence we assessed the potential of re-infection by Delta, Delta AY.1 and B.1 in COVID-19 recovered hamsters after 3 months of infection. Re-infection with Delta and B.1 variants in hamsters showed reduced viral shedding, lung pathology and lung viral load, whereas the upper respiratory tract viral load remained similar to that of first infection. The reduction in viral load and lung pathology after re-infection with Delta AY.1 variant was not marked. Further we assessed the disease characteristics of Delta AY.1 to understand whether it has any replication advantage over Delta variant and B.1 variant, an early isolate in Syrian hamsters. Body weight changes, viral load in respiratory organs, lung pathology, cytokine response and neutralizing antibody response were assessed. Delta AY.1 variant produced milder disease in comparison to Delta variant and the neutralizing response was similar against Delta, B.1 and B.1.351 variant in contrast to Delta or B.1 infected hamsters which showed a significant reduction in neutralization titres against B.1.351. Elevation of IL-6 levels was observed post infection in hamsters after primary infection. The prior infection could not produce sterilizing immunity but the protective effect was evident following reinfection. This indicates the importance of the transmission prevention efforts even after achieving herd immunity.

Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSecondary infections with Delta variant are being widely reported and there are reports of increased disease severity. Delta sub lineages with K417N substitution has caused concern worldwide due to the presence of the same substitution in Beta variant, a Variant of Concern known for its immune evasion. The information on the biological characteristics of this sub lineage is also scanty.

Added value of this studyThe present study showed that the secondary infection with Delta variant does not show any evidence of increased disease severity in hamster model. Delta AY. 1 variant produces mild disease in Syrian hamsters in contrast to severe disease caused by Delta variant. Delta, B.1 and AY.1 variant infected hamster sera showed comparable cross neutralizing response against each other. In contrast to the lower neutralizing response shown by B.1 and Delta variant infected animals against B.1.351 variant, Delta AY.1 showed comparable response as that with other variants.

Implications of the available evidenceSARS-CoV-2 infections do not produce sterilizing immunity but protect from developing severe disease in case of Delta variant re-infection indicating the importance of the transmission prevention efforts even after achieving herd immunity. Delta AY. 1 infection in hamsters did not show any evidence of speculated immune evasion.

category: molecular biology


title: In silico study on the effects of disulfide bonds in ORF8 of SARS-CoV-2

abstract: The COVID-19 epidemic, caused by virus SARS-CoV-2, has been a pandemic and threatening everyones health in the past two years. In SARS-CoV-2, the accessory protein ORF8 plays an important role in immune modulation. Here we present an in silico study on the effects of the disulfide bonds in ORF8, including the effects on the structures, the binding sites and free energy when ORF8 binds to the human leukocyte antigen (HLA-A). Using the explicit solvent Molecular Dynamics (MD) simulations, we collect the conformational ensembles on ORF8 with different disulfide bonds reduction schemes. With a new visualization technique on the local geometry, we analyze the effects of the disulfide bonds on the structure of ORF8. We find that the disulfide bonds have large influences on the loop regions of the surface. Moreover, by performing docking between HLA-A and the conformational ensembles of ORF8, we predict the preferred binding sites and find that most of them are little affected by the disulfide bonds.Further, we estimate the binding free energy between HLA-A and ORF8 with different disulfide bonds reductions. In the end, from the comparison with the available experimental results on the epitopes of ORF8, we validated our binding sites prediction. All the above observations may provide inspirations on inhibitor/drug design against ORF8 based on the binding pathway with HLA-A.

category: biophysics


title: Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants

abstract: The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used naive phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

category: synthetic biology


title: A human monoclonal antibody potently pan-neutralizes SARS-CoV-2 VOCs by targeting RBD invariant sites

abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.

category: immunology


title: A potent human monoclonal antibody with pan-neutralizing activities directly dislocates S trimer of SARS-CoV-2 through binding both up and down forms of RBD


title: Heterologous Vaccination with SARS-CoV-2 Spike saRNA Prime followed by DNA Dual-Antigen Boost Induces Robust Antibody and T-Cell Immunogenicity against both Wild Type and Delta Spike as well as Nucleocapsid Antigens

abstract: We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (SASA S) delivered by a nano-lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression. The N antigen is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the SASA S vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to increase cross-reactivity across variants. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen including the SASA S vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AdS+N boost of an SASA S prime particularly enhanced both CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving SASA S homologous or heterologous vaccination were found to be highly neutralizing of all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strain. The findings here support the clinical testing of heterologous vaccination by an SASA S > AdS+N regimen to provide increased protection against emerging SARS-CoV-2 variants.


title: Heteologous saRNA-Prime, DNA Dual-Antigen-Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies


title: Developing an Antiviral Peptides Predictor with Generative Adversarial Network Data Augmentation

abstract:
MotivationNew antiviral drugs are urgently needed because of emerging viral pathogens increasing severity and drug resistance. Antiviral peptides (AVPs) have multiple antiviral properties and are appealing candidates for antiviral drug development. We developed a sequence-based binary classifier to identify whether an unknown short peptide has AVP activity. We collected AVP sequence data from six existing databases. We used a generative adversarial network to augment the number of AVPs in the positive training dataset and allow our deep convolutional neural network model to train on more data.

ResultsOur classifier achieved outstanding performance on the testing dataset compared with other state-of-the-art classifiers. We deployed our trained classifier on a user-friendly web server.

Availability and implementationAI4AVP is freely accessible at http://axp.iis.sinica.edu.tw/AI4AVP/

Contactcylin@iis.sinica.edu.tw

Supplementary informationSupplementary data is also available.

category: microbiology


title: Copper nanoparticles as modulator of active bacterial population and their physiology in ecosphere

abstract: The widespread use of nanoparticles (NPs) in industrial and consumer products has resulted in their emergence as significant environmental contaminants that can potentially modulate the role of bacteria in environment. This study examines the impact of different sizes of Copper nanoparticles (CuNPs) on the population and physiology of environmentally relevant gram positive (Bacillus) and gam negative (Alcaligenes, and Pseudomonas) bacteria. In general, exposure to CuNPs resulted in 4 to >6 log inactivation in bacterial population. More specifically, after 2hr exposure of Alcaligenes and Pseudomonas to 50 CuNPs, 5.75 and 6.64 log reduction noted, respectively; and their exposure to 100 CuNPs resulted in 5.97 and 6.58 log reduction, respectively. A similar exposure of laboratory and environmental isolates of Bacillus to 50 and 100 CuNPs resulted in 4.84, 4.16 and 4.35, 3.61 log reduction, correspondingly. The exposure induced elicitation of different toxicity pathways in the test bacteria. Bacterial exposure to 50 CuNPs resulted in elevated levels of LDH in Pseudomonas, in contrast these levels decreased in Alcaligenes and Bacillus. Our toxicity studies showed that exposure to CuNP can have various levels of metabolic and cellular modulation in bacterial species, suggesting that the presence of CuNPs in environment can potentially impact the pollutants-attenuation-role of bacteria in environments such as wastewater biological treatment processes.


title: The processing of staphylococcal biofilm images from Fluorescence and Confocal Microscopy to assess in vitro efficacy of antiseptic molecules

abstract: The in vitro efficacy of locally applied antiseptic molecules against staphylococcal biofilm is frequently assessed by a set of standard quantitative and semi-quantitative methods. The development of software for parametric image processing allowed to obtain parametric data also from microscopic images of biofilm dyed with a variety of dyes, especially with propidium iodine and SYTO-9, differentiating dead from live cells. In this work, using confocal/epifluorescent microscopy, we analyzed such major properties of staphylococcal biofilm in vitro as its thickness, cellular density and share of Live/Dead cells within its individual parts. We also scrutinized the impact of sample preparation and antiseptic introduction on the outcome obtained. As a result of our analyses we developed a revelatory method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, in which the microscopic images are processed with the use of ABE formula (Antiseptics Biofilm Eradication) which implements all the data and phenomena detected and revealed within the course of this study. We tested ABE with regard to polyhexanide, pov idone-iodine and hypochlorous antiseptics and found a high correlation between this parameter and the results obtained by means of traditional techniques. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool providing reliable, parametric data with this regard.


title: SARS-CoV-2 Virus like Particles produced by a single recombinant baculovirus generate potent neutralizing antibody that protects against variant challenge

abstract: The Covid-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for the rapid generation of efficient vaccines for emerging disease. Virus-like particles, VLPs, are an established vaccine technology that produces virus-like mimics, based on expression of the structural proteins of a target virus that can stimulate strong neutralizing antibody responses. SARS-CoV-2 is a coronavirus where the basis of VLP formation has been shown to be the co-expression of the spike, membrane and envelope structural proteins. Here we describe the generation of SARS-CoV-2 VLPs by the co-expression of the salient structural proteins in insect cells using the established baculovirus expression system. VLPs were heterologous [~]100nm diameter enveloped particles with a distinct fringe that reacted strongly with SARS-CoV-2 convalescent sera. In a Syrian hamster challenge model, a non-adjuvanted VLPs induced neutralizing antibodies to the VLP-associated Wuhan S protein, reduced virus shedding following a virulent challenge with SARS-CoV-2 (B.1.1.7 variant) and protected against disease associated weight loss. Immunized animals showed reduced lung pathology and lower challenge virus replication than the non-immunized controls. Our data, using an established and scalable technology, suggest SARS-CoV-2 VLPs offer an efficient vaccine that mitigates against virus load and prevents severe disease.


title: Functional analysis of the polar amino acid in TatA transmembrane helix

abstract: The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (pmf) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different pmf components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

category: biochemistry


title: Structural characterization of a MAPR-related archaeal cytochrome b5M protein

abstract: We recently reported that the membrane associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b5 (cytb5) domain proteins, called cytb5M (MAPR-like). Relative to classical cytb5 proteins, MAPR and ctyb5M proteins shared unique sequence elements and a distinct heme binding orientation at an approximately 90 rotation relative to classical cytb5, as demonstrated in the archetypal crystal structure of a cytb5M protein (PDB accession number 6NZX). Here, we present the second crystal structure of an archaeal cytb5M domain (Methanococcoides burtonii WP_011499504.1, PDB:6VZ6). It exhibits similar heme-binding to the 6NZX cytb5M, supporting the deduction that MAPR-like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene.


title: Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

abstract: Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.


title: Th1, Th2 and Th17 inflammatory pathways synergistically correlate with cardiometabolic processes. A case study in COVID-19.

abstract: A predominant source of complication in SARS-CoV-2 patients arises from the cytokine storm, an elevated expression of inflammatory helper T-cell associated cytokines that can lead to tissue damage and organ failure. The high inflammatory burden of this viral infection often results in cardiovascular comorbidities. A better understanding of the interaction between the cytokine storm and cardiovascular proteins might inform medical decisions and therapeutic approaches. We hypothesized that all major helper T-cell inflammatory pathways (Th1, Th2 and Th17) synergistically contribute to cardiometabolic modifications in serum of COVID-19 patients. We proved our hypothesis by integrating Th1, Th2 and Th17 cytokines to predict expression of cardiometabolic proteins profiled by OLINK proteomics.

category: bioinformatics


title: SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens

abstract:
The baseline composition of T cells directly impacts later response to a pathogen, but the complexity of precursor states remains poorly defined. Here we examined the baseline state of SARS-CoV-2 specific T cells in unexposed individuals. SARS-CoV-2 specific CD4+ T cells were identified in pre-pandemic blood samples by class II peptide-MHC tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2 specific T cells that expressed memory phenotype markers, including memory cells with gut homing receptors. T cell clones generated from tetramer-labeled cells cross-reacted with bacterial peptides and responded to stool lysates in a MHC-dependent manner. Integrated phenotypic analyses revealed additional precursor diversity that included T cells with distinct polarized states and trafficking potential to other barrier tissues. Our findings illustrate a complex pre-existing memory pool poised for immunologic challenges and implicate non-infectious stimuli from commensal colonization as a factor that shapes pre-existing immunity.

One Sentence SummaryPre-existing immunity to SARS-CoV-2 contains a complex pool of precursor lymphocytes that include differentiated cells with broad tissue tropism and the potential to cross-react with commensal antigens.


title: Site-specificity of streptococci in the oral microbiome

abstract: Patterns of microbial distribution are determined by as-yet poorly understood rules governing where microbes can grow and thrive. Therefore, a detailed understanding of where bacteria localize is necessary to advance microbial ecology and microbiome-based therapeutics. The site-specialist hypothesis predicts that most microbes in the human oral cavity have a primary habitat within the mouth where they are most abundant. We asked whether this hypothesis accurately describes the distribution of the members of the genus Streptococcus, a clinically relevant taxon that dominates most oral sites. Prior analysis of 16S rRNA gene sequencing data indicated that some oral Streptococcus clades are site-specialists while others may be generalists. However, within complex microbial populations composed of numerous closely-related species and strains, such as the oral streptococci, genome-scale analysis is necessary to provide the resolution to discriminate closely related taxa with distinct functional roles. Here we assess whether individual species within this genus are generalists using publicly available genomic sequence data that provides species-level resolution. We chose a set of high-quality representative genomes for Streptococcus species from the human oral microbiome. Onto these genomes, we mapped short-read metagenomic sequences from supragingival plaque, tongue dorsum, and other sites in the oral cavity. We found that every reliably detectable Streptococcus species in the human oral cavity was a site-specialist and that even closely related species such as S. mitis, S. oralis, and S. infantis specialized in different sites. These findings indicate that closely related bacteria can have distinct habitat distributions in the absence of dispersal limitation and under similar environmental conditions and immune regimes. These three species also share substantially the same species-specific core genes indicating that neither taxonomy nor gene content are clear predictors of site-specialization. Site-specificity may instead be influenced by subtle characteristics such as nucleotide-level divergences within conserved genes.


title: A bistable orthogonal prokaryotic differentiation system underlying development of conjugative transfer competence

abstract: The mechanisms and impact of horizontal gene transfer processes to distribute gene functions with potential adaptive benefit among prokaryotes have been well documented. In contrast, little is known about the life-style of mobile elements mediating horizontal gene transfer, whereas this is the ultimate determinant for their transfer fitness. Here, we investigate the life-style of an integrative and conjugative element (ICE) within the genus Pseudomonas that stands model for a widespread family transmitting genes for xenobiotic compound metabolism and antibiotic resistances. The ICE only transfers from a small fraction of cells in a population, which we uncover here, results from a dedicated transfer competence program imposed by the ICE. Transfer competence is orthogonally maintained in individual cells in which it is activated, making them the centerpiece of ICE conjugation. The components mediating transfer competence are widely conserved, underscoring their selected fitness for efficient transfer of this class of mobile elements.


title: Communication determines population-level fitness under cation stress by modulating the ratio of motile to sessile B. subtilis cells

abstract: Bacterial populations frequently encounter potentially lethal environmental stress factors. Growing Bacillus subtilis populations are comprised of a mixture of "motile" and "sessile" cells but how this affects population-level fitness under stress is poorly understood. Here, we show that, unlike sessile cells, motile cells are readily killed by monovalent cations under conditions of nutrient deprivation - owing to elevated expression of the lytABC operon, which codes for a cell-wall lytic complex. Forced induction of the operon in sessile cells also causes lysis. We demonstrate that population composition is regulated by the quorum sensing regulator ComA, which can favor either the motile or the sessile state. Specifically social interactions by ComX-pheromone signaling enhance population-level fitness under stress. Our study highlights the importance of characterizing population composition and cellular properties for studies of bacterial physiology and functional genomics. Our findings open new perspectives for understanding the functions of autolysins and collective behaviors that are coordinated by chemical and electrical signals, with implications for multicellular development and biotechnology.


title: Safety, Immunogenicity and Efficacy of a Recombinant Vesicular Stomatitis Virus Vectored Vaccine Against Severe Fever with Thrombocytopenia Syndrome and Heartland Bandaviruses

abstract:
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a recently emerged tickborne virus in east Asia with over 8,000 confirmed cases. With a high case fatality ratio, SFTSV has been designated a high priority pathogen by the WHO and the NIAID. Despite this, there are currently no approved therapies or vaccines to treat or prevent SFTS. Vesicular stomatitis virus (VSV) represents an FDA-approved vaccine platform that has been considered for numerous viruses due to its low sero-prevalence in humans, ease in genetic manipulation and promiscuity in incorporating foreign glycoproteins into its virions. In this study, we developed a recombinant VSV (rVSV) expressing the SFTSV glycoproteins Gn/Gc (rVSV-SFTSV) and assessed its safety, immunogenicity and efficacy in mice. We demonstrate that rVSV-SFTSV is safe when given to immunocompromised animals and is not neuropathogenic when injected intracranially into young immunocompetent mice. Immunization of Ifnar-/- mice with rVSV-SFTSV resulted in high levels of neutralizing antibodies and protection against lethal SFTSV challenge. Additionally, passive transfer of sera from immunized Ifnar-/- mice into naive animals was protective when given pre- or post-exposure. Finally, we demonstrate that immunization with rVSV-SFTSV cross protects mice against challenge with the closely related Heartland virus despite low neutralizing titers to the virus. Taken together, these data suggest that rVSV-SFTSV is a promising vaccine candidate.

ImportanceTick borne diseases are a growing threat to human health. Severe fever with thrombocytopenia syndrome (SFTS) and Heartland viruses are recently recognized, highly-pathogenic, tick-transmitted viruses. The fatality rates for individuals infected with SFTSV or HRTV are high and there are no therapeutics or vaccines available. The recent introduction of the tick vector for SFTSV (Haemaphysalis longicornis) to the eastern half of the United States and Austrailia raises concerns for SFTSV outbreaks outside East Asia. Here we report the development of a potential vaccine for SFTSV and HRTV based on the viral vector platform that has been successfully used for an Ebola vaccine. We demonstrate that the rVSV-SFTSV protects from lethal SFTSV or HRTV challenge when given as a single dose. We evaluated possible pathogenic effects of the vaccine and show that it is safe in immune compromised animlas and when introduced into the central nervous system.


title: Structure and conservation of amyloid spines from the Candida albicans Als5 adhesin including similarity to human LARKS

abstract:
Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation and fungal-bacterial co-aggregation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a [~]300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-{beta} spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked {beta}-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-{beta} structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins. Thus, cross-{beta} structures are often involved in fungal pathogenesis and potentially in antifungal therapy.

ImportanceFungal adhesins form cell-to-cell bonds in biofilms. Many of the cellular interactions are dependent on formation of amyloid-like cross-{beta} protein aggregates. Such structures are called  functional amyloids because they perform physiological activities and they are dependent on the same types of protein interactions that form the more familiar amyloid deposits in neurodegenerative diseases. We have identified sequence segments that form cross-{beta} structures in the Als5 adhesin from the human pathogen Candida albicans. Such sequences are widespread among ALS family adhesins, including those from other human pathogens including Candida auris. Moreover, we revealed a structural similarity in a segment originating from Als5 threonine-rich low complexity region to human LARKS, pointing on a common structural motif coding for functional amyloids in different kingdoms of life.


title: Plasmodiophora brassicae in Mexico, from anecdote to fact

abstract: For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been given by granted. However, after a long search in the scientific literature in English and Spanish, as well as grey literature including thesis and government reports, we were not able to find any information regarding the actual detection of the pathogen, hosts affected, areas with the disease, or any real information about clubroot ( hernia de la col, in Mexico). To confirm if P. brassicae was indeed in Mexico, we started a true detective adventure. First, we identified agricultural communities in south-east Mexico known to grow cruciferous crops. Second, we asked to the growers if they have ever seen clubroot symptoms, showing them during the inquires pictures of the characteristic galls that might have been present in their crops. Third, we collected soil from two of the communities with positive response and grew an array of cruciferous in the soil as baits to "fish" the clubroot pathogen. We detected the presence of galls in the roots of 32 plants and observed the presence of resting spores. Through a P. brassicae specific PCR assay, we were able to confirm the presence of the clubroot pathogen in the samples and in Mexico for the very first time. This study is the first report and identification of P. brassicae in Mexico, opening the doors to understand the genetic diversity of this elusive and devastating plant pathogen.


title: The Atypical Antipsychotic Quetiapine Induces Multiple Antibiotic Resistance in Escherichia coli

abstract: Atypical antipsychotic (AAP) medication is a critical tool for treating symptoms of psychiatric disorders. While AAPs primarily target dopamine (D2) and serotonin (5HT2A and 5HT1A) receptors, they also exhibit intrinsic antimicrobial activity as an off-target effect. Because AAPs are often prescribed to patients for many years, a potential risk associated with long-term AAP use is the unintended emergence of bacteria with antimicrobial resistance (AMR). Here, we show that exposure to the AAP quetiapine at estimated gut concentrations promotes AMR in Escherichia coli after six weeks. Quetiapine-exposed isolates exhibited an increase in minimal inhibitory concentrations (MICs) for ampicillin, tetracycline, ceftriaxone, and levofloxacin. By whole genome sequencing analysis, we identified mutations in genes that confer AMR, including the repressor for the multiple antibiotic resistance mar operon (marR), and real-time RT-qPCR analysis showed increased levels of marA, acrA, and tolC mRNAs and a reduced level of ompF mRNA in the isolates carrying marR mutations. To determine the contribution of each marR mutation to AMR, we constructed isogenic strains carrying individual mutant marR alleles in the parent background and re-evaluated their resistant phenotypes using MIC and RT-qPCR assays. While marR mutations induced a robust activity of the mar operon, they resulted in only a modest increase in MICs. Interestingly, although these marR mutations did not fully recapitulate the AMR phenotype of the quetiapine-exposed isolates, we show that marR mutations promote growth fitness in the presence of quetiapine. Our findings revealed an important link between the use of AAPs and AMR development in E. coli.


title: The decrotonylase FoSir5 facilitates mitochondrial metabolic state switching in conidial germination of Fusarium oxysporum

abstract: Fusarium oxysporum is one of the most important pathogenic fungi with a broad range of plant and animal hosts. The first key step of its infection cycle is conidial germination, but there is limited information available on the molecular events supporting this process. We show here that germination is accompanied by a sharp decrease in expression of FoSir5, an ortholog of the human lysine deacetylase SIRT5. We observe that FoSir5 decrotonylates a subunit of the fungal pyruvate dehydrogenase complex (FoDLAT) at K148, resulting in inhibition of the activity of the complex in mitochondria. Moreover, FoSir5 decrotonylates histone H3K18, leading to a downregulation of transcripts encoding enzymes of aerobic respiration pathways. Thus, the activity of FoSir5 coordinates regulation in different organelles to steer metabolic flux through respiration. As ATP content is positively related to fungal germination, we propose that FoSir5 negatively modulates conidial germination in F. oxysporum through its metabolic impact. These findings provide insights into the multifaceted roles of decrotonylation, catalysed by FoSir5, that support conidial germination in F. oxysporum.


title: TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

abstract: Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. Conversely, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.


title: Topoisomerases I and II facilitate condensin DC translocation to organize and repress X chromosomes in C. elegans

abstract: Condensin complexes are evolutionarily conserved molecular motors that translocate along DNA and form loops. While condensin-mediated DNA looping is thought to direct the chain-passing activity of topoisomerase II to separate sister chromatids, it is not known if topological constraints in turn regulate loop formation in vivo. Here we applied auxin inducible degradation of topoisomerases I and II to determine how DNA topology affects the translocation of an X chromosome specific condensin that represses transcription for dosage compensation in C. elegans (condensin DC). We found that both topoisomerases colocalize with condensin DC and control its movement at different genomic scales. TOP-2 depletion hindered condensin DC translocation over long distances, resulting in accumulation around its X-specific recruitment sites and shorter Hi-C interactions. In contrast, TOP-1 depletion did not affect long-range spreading but resulted in accumulation of condensin DC within expressed gene bodies. Both TOP-1 and TOP-2 depletions resulted in X chromosome transcriptional upregulation indicating that condensin DC translocation at both scales is required for its function in gene repression. Together the distinct effects of TOP-1 and TOP-2 on condensin DC distribution revealed two distinct modes of condensin DC association with chromatin: long-range translocation that requires decatenation/unknotting of DNA and short-range translocation across genes that requires resolution of transcription-induced supercoiling.


title: RNA-guided cell targeting with CRISPR/RfxCas13d collateral activity in human cells

abstract: While single-cell sequencing has allowed rapid identification of novel cell types or states and associated RNA markers, functional studies remain challenging due to the lack of tools that are able to target specific cells based on these markers. Here we show that targeting a single marker RNA with CRISPR/RfxCas13d led to collateral transcriptome destruction in human cells, which can be harnessed to inhibit cell proliferation or to suppress cell state transition.


title: A minimal model demonstrates the control of mouse sperm capacitation by a positive feedback loop between sNHE and SLO3

abstract: When mammalian spermatozoa are released in the female reproductive tract, they are incapable of fertilizing the oocyte. They need a prolonged exposure to the alkaline medium of the female genital tract before their flagellum gets hyperactivated and the acrosome reaction can take place, allowing the sperm to interact with the oocyte. Ionic fluxes across the sperm membrane are involved in two essential aspects of capacitation: the increase in intracellular pH and the membrane hyperpolarization. In particular, it has been shown that the SLO3 potassium channel and the sNHE sodium-proton transporter, two sperm-specific transmembrane proteins, are necessary for the capacitation process to occur. As the SLO3 channel is activated by an increase in intracellular pH and sNHE is activated by hyperpolarization, they act together as a positive feedback system. Mathematical modeling provides a unique tool to capture the essence of a molecular mechanism and can be used to derive insight from the existing data. We have therefore developed a theoretical model formalizing the positive feedback loop between SLO3 and sHNE in mouse epididymal sperm to see if this non-linear interaction can provide the core mechanism explaining the existence of uncapacited and capacitated states. We show that the proposed model can fully explain the switch between the uncapacitated and capacited states and also predicts the existence of a bistable behaviour. Furthermore, our model indicates that SLO3 inhibition, above a certain threshold, is effective to completely abolish capacitation.

category: cell biology


title: Protein proximity networks and functional evaluation of the Casein Kinase 1 γ family reveals unique roles for CK1γ3 in WNT signaling

abstract: The WNT/{beta}-catenin signaling pathway is evolutionarily conserved and controls normal embryonic development, adult tissue homeostasis and regeneration. Aberrant activation or suppression of WNT signaling contributes to cancer initiation and progression, developmental disorders, neurodegeneration, and bone disease. Despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and  dark. Here we studied the function of the CSNK1{gamma} subfamily of human kinases. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1{gamma}1, CSNK1{gamma}2, and CSNK1{gamma}3 revealed numerous established components of the {beta}-catenin- dependent and independent WNT signaling pathway, as well as novel interactors. In gain-of- function experiments leveraging a panel of transcriptional reporters, CSNK1{gamma}3 but not CSNK1{gamma}1 or CSNK1{gamma}2 activated {beta}-catenin-dependent WNT signaling and the Notch pathway. Within the family, CSNK1{gamma}3 expression uniquely induced LRP6 phosphorylation. Conversely, siRNA- mediated silencing of CSNK1{gamma}3 alone had no impact on WNT signaling, though co-silencing of all three family members decreased WNT pathway activity. We characterized two moderately selective and potent small molecule inhibitors of the CSNK1{gamma} family. These inhibitors and a CSNK1{gamma}3 kinase dead mutant suppressed but did not eliminate WNT-driven LRP6 phosphorylation and {beta}-catenin stabilization. Our data suggest that while CSNK1{gamma}3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.


title: Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation

abstract: Replication of the human genome initiates within broad zones of ~ 150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq) based on density substitution. Newly-replicated DNA is rendered  heavy-light (HL) by incorporation of BrdUTP, unreplicated DNA remaining  light-light (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.

category: genomics


title: Gut transcriptome reveals differential gene expression and enriched pathways linked to immune activation in response to weaning in pigs.

abstract:
Weaning represents one of the most critical periods in pig production associated with increase in disease risk, reduction in performance and economic loss. Physiological changes faced by piglets during the weaning period have been well characterised, however little is currently known about the underlying molecular pathways involved in these processes. As pig meat remains one of the most consumed sources of protein worldwide, understanding how these changes are mediated is critical to improve pig production and consequently sustainable food production globally. In this study, we evaluated the effect of weaning on transcriptomic changes in the colon of healthy piglets over time using an RNA-sequencing approach.

The findings revealed a complex and coordinated response to weaning with the majority of genes found to be rapidly differentially expressed within one day post weaning. Multiple genes and pathways affected by weaning in the colon were associated with immune regulation, cell signalling and bacterial defence. NOD-like receptors, Toll-like receptor and JAK-STAT signalling pathways were amongst the pathways significantly enriched. Immune activation was evidenced by the enrichment of pathways involved in interferon response, cytokines interactions, oxidoreductase activities and response to microbial invasion. Biosynthesis of amino acids, in particular arginine, was also amongst the most enriched KEGG pathways in weaned pigs, reinforcing the critical role of arginine in gut homeostasis under stress conditions.

Overall, transcriptomic and physiological results suggest that pigs going through the weaning transition undergo a transient period of inflammatory state with a temporary breakdown of barrier functions in the gut. These findings could provide valuable tools to monitor host response post weaning, and may be of particular relevance for the investigation and development of intervention strategies aimed to reduce antibiotic use and improve pig health and performance.


title: Rolling Circle RNA Synthesis Catalysed by RNA

abstract: RNA-catalysed RNA replication is widely considered a key step in the emergence of lifes first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5 and 3 ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.


title: A shape-shifting nuclease unravels structured RNA

abstract: RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts that may otherwise interfere with cellular programs. The enzyme Dis3-like protein 2 (Dis3L2) is a 3-5 exoribonuclease that, through its RNA turnover activity, plays a critical role in human development1. Dis3L2 can independently degrade structured substrates and its targets include many coding and non-coding 3-uridylated RNAs1-5. While the basis for Dis3L2s substrate recognition has been well-characterized6, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining electron cryo-microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for single-stranded (ss) and double-stranded (ds) RNA processing. We discovered a dramatic conformational change that is triggered by the dsRNA, involving repositioning of two cold shock domains by 70 [A]. This movement exposes a trihelix-linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not ssRNA, degradation. These findings reveal the conformational plasticity of this enzyme, and detail a novel mechanism of structured RNA degradation.


title: Functional classification and validation of yeast prenylation motifs using machine learning and genetic reporters

abstract: Protein prenylation by farnesyltransferase (FTase) is often described as the targeting of a cysteine-containing motif (CaaX) that is enriched for aliphatic amino acids at the a1 and a2 positions, while quite flexible at the X position. Prenylation prediction methods often rely on these features despite emerging evidence that FTase has broader target specificity than previously considered. Using a machine learning approach and training sets based on canonical (prenylated, proteolyzed, and carboxymethylated) and recently identified shunted motifs (prenylation only), this study aims to improve prenylation predictions with the goal of determining the full scope of prenylation potential among the 8000 possible Cxxx sequence combinations. Further, this study aims to subdivide the prenylated sequences as either shunted (i.e., uncleaved) or cleaved (i.e., canonical). Predictions were determined for Saccharomyces cerevisiae FTase and compared to results derived using currently available prenylation prediction methods. In silico predictions were further evaluated using in vivo methods coupled to two yeast reporters, the yeast mating pheromone a-factor and Hsp40 Ydj1p, that represent proteins with canonical and shunted CaaX motifs, respectively. Our machine learning based approach expands the repertoire of predicted FTase targets and provides a framework for functional classification.


title: Development of PRPK Directed Phthalimides

abstract: Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies.


title: Dihydroartemisinin broke immune evasion through YAP1/JAK1/STAT1, 3 pathways to enhance anti-PD-1 therapy in hepatocellular carcinoma

abstract: The efficacy of anti-PD-1 therapy is not as expected in patients with hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP1) was overexpressed and activated in HCC. This study aimed to investigate the potential mechanism and inhibitor of YAP1 on immune evasion, and promote anti-PD-1 therapy in HCC. Here, we showed that dihydroartemisinin (DHA), an FDA approved drug, directly suppressed YAP1 expression, leading to break immune evasion in liver tumor niche, characterized by decreased PD-L1 in liver tumor cells and increased CD8+ T cell infiltration. Mechanismly, YAP1 is not only directly related to PD-L1, but also involved in activating the JAK1/STAT1, 3 pathways. Moreover, Yap1 knockout elevated CD4+ and CD8+ T cells in liver tumor niche of Yap1LKO mice. Consistently, verteporfin, YAP1 inhibitor, decreased TGF-{beta} in liver tumor niche and exhausted CD8+ T cells in spleen. Furthermore, DHA combined with anti-PD-1 treatment promoted CD4+ T cell infiltration in the spleen and CD8+ T cells in tumor tissues. Thus, we provide a new combined therapeutic strategy for anti-PD-1 with DHA, a potent YAP1 inhibitor, in HCC.

category: cancer biology


title: Replication Study: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

abstract:
As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Bhargava et al., 2016) that described how we intended to replicate selected experiments from the paper "RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth" (Hatzivassiliou et al., 2010). Here we report the results. We found two unrelated RAF inhibitors, PLX4720 or GDC-0879, selectively inhibited BRAF(V600E) cell proliferation, while the MEK inhibitor, PD0325901, inhibited BRAF(V600E), wild-type RAF/RAS, and mutant RAS cancer cell proliferation, similar to the original study (Figure 1A; Hatzivassiliou et al., 2010). We found knockdown of CRAF, but not BRAF, in mutant RAS cells attenuated the phospho-MEK induction observed after PLX4720 treatment, similar to the original study (Figure 2B; Hatzivassiliou et al., 2010). The original study reported analogous results with GDC-0879, which was not observed in this replication, although unexpected control results confound the interpretation. We also attempted a replication of an assay with recombinant proteins to test the differential effect of RAF inhibitors on BRAF-CRAF heterodimerization (Figure 4A; Hatzivassiliou et al., 2010). Although we were unable to conduct the experiment as planned, we observed differential binding of BRAF by RAF inhibitors; however, it was between BRAF and beads, independent of CRAF. While these data were unable to address whether, under the conditions of the original study, the same observations could be observed, we discuss key differences between the original study and this replication that are important to consider for further experiments. Finally, where possible, we report meta-analyses for each result.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/470372v2_fig1.gif" ALT="Figure 1">
View larger version (7K):
org.highwire.dtl.DTLVardef@1f77d0eorg.highwire.dtl.DTLVardef@1333494org.highwire.dtl.DTLVardef@c10824org.highwire.dtl.DTLVardef@fda30f_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1.C_FLOATNO Cellular dose response curves for RAF and MEK inhibitors in BRAF(V600E), mutant RAS, and wild-type RAF/RAS cell lines Cell viability assays were performed for RAF inhibitors (PLX4720 and GDC-0879) and MEK inhibitor (PD0325901) against A375, (BRAF(V600E)), HCT116 (mutant RAS), and MeWo (wild-type RAF/RAS) cells. Absolute half-maximum effective concentration (EC50) values ({micro}M) were determined for each biological repeat. EC50 values unable to be accurately estimated are reported as either >20 {micro}M or <0.078 {micro}M, which are the largest and smallest doses tested, respectively. Absolute EC50 values for each biological repeat for A375, HCT116, and MeWo cells treated with (A) PLX4720, (B) GDC-0879, or (C) PD0325901 for this replication attempt are plotted with the EC50 value reported in Hatzivassiliou et al. (2010) displayed as a single point (red triangle) for comparison. Where possible the mean and 95% confidence interval of the replication data are shown. Additional details for this experiment can be found at https://osf.io/52zp9/.

C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/470372v2_fig2.gif" ALT="Figure 2">
View larger version (34K):
org.highwire.dtl.DTLVardef@1dc3058org.highwire.dtl.DTLVardef@e9f5d9org.highwire.dtl.DTLVardef@baf5eorg.highwire.dtl.DTLVardef@126717a_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 2.C_FLOATNO Cellular dose response curves for each biological repeat This is the same experiment as in Figure 1. The dose response curve of each biological repeat [n=4] for A375, HCT116, and MeWo cells treated with (A) PLX4720, (B) GDC-0879, or (C) PD0325901 for this replication attempt are plotted. Additional details for this experiment can be found at https://osf.io/52zp9/.

C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/470372v2_fig4.gif" ALT="Figure 4">
View larger version (33K):
org.highwire.dtl.DTLVardef@e4357eorg.highwire.dtl.DTLVardef@73e9ceorg.highwire.dtl.DTLVardef@1fb01c6org.highwire.dtl.DTLVardef@31a799_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 4.C_FLOATNO Testing of BRAF and CRAF antibodies used in original study The BRAF and CRAF antibodies reported in the original study (Hatzivassiliou et al., 2010) were tested for suitability by Western blot. (A) Representative Western blots and Ponceau-stained membrane (experiment performed independently 3 times) of 0.5 {micro}g of purified recombinant BRAF kinase domain (KD). Membranes were probed with the BRAF antibody used in the original study (RRID:AB_1840330) or with a different BRAF antibody used in this replication attempt (RRID:AB_2721113). (B) Representative Western blots and Ponceau-stained membrane (experiment performed independently 3 times) of 0.5 {micro}g of purified recombinant GST tagged CRAF KD. Membranes were probed with the CRAF antibody used in the original study (RRID:AB_397552) or with a different CRAF antibody used in this replication attempt (RRID:AB_10989977). (C) Representative Western blots (experiment performed once) of 16 {micro}g of whole cell lysates from the indicated human cell lines. Membranes were probed with the BRAF (RRID:AB_1840330) or CRAF (RRID:AB_397552) antibodies used in the original study. (D) Screenshot from the Constraint-based Multiple Alignment Tool (COBALT) (Papadopoulos and Agarwala, 2007) showing the alignment of full-length BRAF (NCBI Reference Sequence: NP_004324.2), the region of BRAF used as the immunogenic peptide used to generate the BRAF antibody used in the original study (RRID:AB_1840330), the BRAF KD used in this replication attempt, and the BRAF KD used in the original study. (E) Screenshot from COBALT showing the alignment of full-length CRAF (NCBI Reference Sequence: NP_002871.1), the region of CRAF used as the immunogenic peptide used to generate the CRAF antibody used in the original study (RRID:AB_397552), and the CRAF KD used in the original study and this replication attempt. Additional details for this experiment can be found at https://osf.io/6f3sk/.

C_FIG


title: Endogenous giant viruses shape intraspecies genomic variability in the model green alga Chlamydomonas reinhardtii

abstract: Chlamydomonas reinhardtii is an important eukaryotic alga that has been studied as a model organism for decades. Despite extensive history as a model system, phylogenetic and genetic characteristics of viruses infecting this alga have remained elusive. We analyzed high-throughput genome sequence data of C. reinhardtii field isolates, and in six we discovered sequences belonging to endogenous giant viruses that reach up to several hundred kilobases in length. In addition, we have also discovered the entire genome of a closely related giant virus that is endogenized within the genome of Chlamydomonas incerta, the closest sequenced phylogenetic relatives of C. reinhardtii. Endogenous giant viruses add hundreds of new gene families to the host strains, highlighting their contribution to the pangenome dynamics and inter-strain genomic variability of C. reinhardtii. Our findings suggest that the endogenization of giant viruses can have important implications for structuring the population dynamics and ecology of protists in the environment.


title: Rapid and efficient adaptation of the dTAG system in mammalian development reveals stage specific requirements of NELF

abstract:
Targeted protein degradation methods offer a unique avenue to assess a proteins function in a variety of model systems. Recently, these approaches have been applied to mammalian cell culture models, enabling unprecedented temporal control of protein function. However, the efficacy of these systems at the tissue and organismal levels in vivo is not well established. Here, we tested the functionality of the degradation tag (dTAG) degron system in mammalian development. We generated a homozygous knock-in mouse with a FKBPF36V tag fused to Negative elongation factor b (Nelfb) locus, a ubiquitously expressed protein regulator of transcription. In the first validation of targeted endogenous protein degradation across mammalian development, we demonstrate that irrespective of the route of administration the dTAG system is safe, rapid, and efficient in embryos from the zygote to midgestation stages. Additionally, acute early depletion of NELFB revealed a specific role in zygote-to-2-cell development and Zygotic Genome Activation (ZGA).

HIGHLIGHTSO_LIgenetically engineered mouse model harboring a FKBPF36V knock-in to evaluate kinetics and efficacy of the dTAG degron system in vivo
C_LIO_LIsystem is non-toxic, and allows acute and efficient degradation of a FKBPF36V- tagged endogenous protein during in utero embryo development
C_LIO_LIsystem nables fine temporal degradation and reversibility of depletion across embryonic stages
C_LIO_LIstage-specific depletion reveals a role for NELFB during mouse ZGA
C_LI

category: developmental biology


title: MaveDB v2: a curated community database with over three million variant effects from multiplexed functional assays

abstract: A central problem in genomics is understanding the effect of individual DNA variants. Multiplexed Assays of Variant Effect (MAVEs) can help address this challenge by measuring all possible single nucleotide variant effects in a gene or regulatory sequence simultaneously. Here we describe MaveDB v2, which has become the database of record for MAVEs. MaveDB now contains a large fraction of published studies, comprising over two hundred datasets and three million variant effect measurements. We created tools and APIs to streamline data submission and access, transforming MaveDB into a hub for the analysis and dissemination of these impactful datasets.


title: Oxytocin promotes social grooming in bonobos: testing the biobehavioural feedback loop hypothesis

abstract: Oxytocin has attracted research attention due to its role in promoting social bonding. One notable recent hypothesis is the biobehavioral feedback loop, which posits that the oxytocin system has evolved to support the formation and maintenance of social bonds through a positive feedback loop, where oxytocin promotes social behaviours which then cause oxytocin release themselves. In the two Pan species, humans closest relatives, oxytocin is known to be released following key behaviours related to social bonding, such as social grooming in chimpanzees and female-female sexual behaviour in bonobos. However, no experimental evidence has demonstrated that oxytocin promotes such socio-positive behaviours. To test this, we administered nebulized oxytocin or saline placebo to a group of female bonobos and subsequently observed the change in their gross behavior during free interaction. We found that bonobos groomed other group members significantly more frequently in the oxytocin compared to placebo condition. Other behavioural measures did not largely differ between conditions, except for a nonsignificant trend for reduction in abnormal regurgitation/reingestion behaviour. Overall, we found that oxytocin promoted socio-positive interaction in bonobos, providing support for the biobehavioural feedback loop hypothesis of oxytocin in bonobo social evolution.

category: animal behavior and cognition


title: Oxytocin promotes social grooming in bonobos


title: monaLisa: an R/Bioconductor package for identifying regulatory motifs

abstract:
Proteins binding to specific nucleotide sequences, such as transcription factors, play key roles in the regulation of gene expression. Their binding can be indirectly observed via associated changes in transcription, chromatin accessibility, DNA methylation and histone modifications. Identifying candidate factors that are responsible for these observed experimental changes is critical to understand the underlying biological processes. Here we present monaLisa, an R/Bioconductor package that implements approaches to identify relevant transcription factors from experimental data. The package can be easily integrated with other Bioconductor packages and enables seamless motif analyses without any software dependencies outside of R.

AvailabilitymonaLisa is implemented in R and available on Bioconductor at https://bioconductor.org/packages/monaLisa with the development version hosted on GitHub at https://github.com/fmicompbio/monaLisa.

Contactmichael.stadler@fmi.ch


title: sciCAN: Single-cell chromatin accessibility and gene expression data integration via Cycle-consistent Adversarial Network

abstract: The booming single-cell technologies bring a surge of high dimensional data that come from different sources and represent cellular systems from different views. With advances in single-cell technologies, integrating single-cell data across modalities arises as a new computational challenge and gains more and more attention within the community. Here, we present a novel adversarial approach, sciCAN, to integrate single-cell chromatin accessibility and gene expression data in an unsupervised manner. We benchmarked sciCAN with 3 state-of-the-art (SOTA) methods in 5 scATAC-seq/scRNA-seq datasets, and we demonstrated that our method dealt with data integration with better balance of mutual transferring between modalities than the other 3 SOTA methods. We further applied sciCAN to 10X Multiome data and confirmed the integrated representation preserves information of the hematopoietic hierarchy. Finally, we investigated CRSIPR-perturbed single-cell K562 ATAC-seq and RNA-seq data to identify cells with related responses to different perturbations in these different modalities.


title: Oxytocin signaling in the posterior hypothalamus prevents hyperphagic obesity in mice

abstract: Decades of studies have revealed molecular and neural circuit bases for body weight homeostasis. Neural hormone oxytocin (OT) has received attention in this context because it is produced by neurons in the paraventricular hypothalamic nucleus (PVH), a known output center of hypothalamic regulation of appetite. OT has an anorexigenic effect, as shown in human studies, and can mediate satiety signals in rodents. However, the function of OT signaling in the physiological regulation of appetite has remained in question, because whole-body knockout (KO) of OT or OT receptor (OTR) has little effect on food intake. We herein show that acute conditional KO (cKO) of OT selectively in the adult PVH, but not in the supraoptic nucleus, markedly increases body weight and food intake, with an elevated level of plasma triglyceride and leptin. Intraperitoneal administration of OT rescues the hyperphagic phenotype of the PVH OT cKO model. Furthermore, we show that cKO of OTR selectively in the posterior hypothalamic regions, which include the primary centers for appetite regulations, phenocopies hyperphagic obesity. Collectively, these data functionally reveal that OT signaling in the posterior hypothalamic regions suppresses excessive food intake.


title: How do spike collisions affect spike sortingperformance?

abstract: Recently, a new generation of devices have been developed to record neural activity simultaneously from hundreds of electrodes with a very high spatial density, both for in vitro and in vivo applications. While these advances enable to record from many more cells, they also dramatically increase the amount overlapping "synchronous" spikes (colliding in space and/or in time), challenging the already complicated process of spike sorting (i.e. extracting isolated single-neuron activity from extracellular signals). In this work, we used synthetic ground-truth recordings to quantitatively benchmark the performance of state-of-the-art spike sorters focusing specifically on spike collisions. Our results show that while modern template-matching based algorithms are more accurate than density-based approaches, all methods, to some extent, failed to detect synchronous spike events of neurons with similar extracellular signals. Interestingly, the performance of the sorters is not largely affected by the the spiking activity in the recordings, with respect to average firing rates and spike-train correlation levels.


title: Dynamics of alpha suppression and enhancement may be related to resource competition in cross-modal cortical regions

abstract: In the face of multiple sensory streams, there may be competition for processing resources in multimodal cortical area devoted to establishing representations. In such cases, alpha oscillations may serve to maintain the relevant representations and protect them from interference, whereas theta oscillations may facilitate their updating when needed. It can be hypothesized that these oscillations would differ in response to an auditory stimulus when the eyes are open or closed, as intermodal resource competition may be more prominent in the former than in the latter case. Across two studies we investigated the role of alpha and theta power in multimodal competition using an auditory task with the eyes open and closed, respectively enabling and disabling visual processing in parallel with the incoming auditory stream. In a passive listening task (Study 1a), we found alpha suppression following a pip tone with both eyes open and closed, but subsequent alpha enhancement only with closed eyes. We replicated this eyes-closed alpha enhancement in an independent sample (Study 1b). In an active auditory oddball task (Study 2), we again observed the eyes open/eyes closed alpha pattern found in Study 1 and also demonstrated that the more attentionally demanding oddball trials elicit the largest oscillatory effects. Theta power did not interact with eye status in either study. We propose a hypothesis to account for the findings in which alpha may be endemic to multimodal cortical areas in addition to visual ones.


title: Micronized curcumin causes hyperlocomotion in zebrafish larvae

abstract: Zebrafish larvae have been widely used in neuroscience and drug research and development. In the larval stage, zebrafish present a broad behavioral repertoire and physiological responses similar to adults. Curcumin (CUR), a major component of Curcuma longa L. (Zingiberaceae), has demonstrated the ability to modulate several neurobiological processes relevant to mental disorders in animal models. However, the low bioavailability of this compound can compromise its in vivo biological potential. Interestingly, it has been shown that micronization can increase the biological effects of several compounds. Thus, in this study, we compared the effects of acute exposure for 30 minutes to the following solutions: water (control), 0.1% DMSO (vehicle), 1 M CUR, or 1 M micronized curcumin (MC) in zebrafish larvae 7 days post-fertilization (dpf). We analyzed locomotor activity (open tank test), anxiety (light/dark test), and avoidance behavior (aversive stimulus test). Moreover, we evaluated parameters of oxidative status (thiobarbituric acid reactive substances and non-protein thiols levels). MC increased the total distance traveled and absolute turn angle in the open tank test. There were no significant differences in the other behavioral or neurochemical outcomes. The increase in locomotion induced by MC may be associated with a stimulant effect on the central nervous system, which was evidenced by the micronization process.


title: Hunchback activates Bicoid in post-mitotic Pair1 neurons to regulate synapse number

abstract: The proper formation and function of neural circuits is crucial for cognition, sensation, and behavior. Neural circuits are highly-specific, and this specificity is dependent on neurons developing key features of their individual identities: morphology, anatomical location, molecular expression and biophysiological properties. Previous research has demonstrated that a neurons identity is, in part, generated by the temporal transcription window the neuron is born in, and the homeodomain transcription factors expressed in the mature neuron. However, whether temporal transcription factors and homeodomain transcription factors regulate neural circuit formation, maintenance and function remains unknown. Here, we utilize a well-characterized neural circuit in the Drosophila larvae, the Pair1 neuron. We determined that in the Pair1 neuron, the temporal transcription factor Hunchback activates the homeodomain transcription factor Bicoid (Bcd). Both Hunchback and Bcd are expressed in Pair1 throughout larval development. Interestingly, Hunchback and Bcd were not required in Pair1 for neurotransmitter identity or axonal morphology, but were required for synapse density. We found that these transcription factors were functioning post-mitotically in Pair1 to regulate synapse density. Additionally, knocking down Hunchback and Bcd in Pair1 neurons disrupted the behavioral output of the circuit. We utilized the genetic tool TransTango to determine that Hunchback function in Pair1 is to repress forming synapses with erroneous neurons. To our knowledge, these data are the first to show Hunchback activating Bcd expression, as well as the first to demonstrate a role for Hunchback and Bcd post-mitotically.


title: Transcriptional adaptation of sensory neurons to GPCR identity and activity

abstract: Sensory adaptation is critical to extract information from a changing world. Taking advantage of the extensive parallel coding lines present in the olfactory system, we explored the potential variations of neuronal identities before and after olfactory experience. We found that at rest, the transcriptomic profiles of olfactory sensory neuron populations are already highly divergent, specific to the olfactory receptor they express, and are surprisingly associated with the sequence of these latter. These divergent profiles further evolve in response to the environment, as odorant exposure leads to massive reprogramming via the modulation of transcription. Adenylyl cyclase 3, but not other main elements of the olfactory transduction cascade, plays a critical role in this activity-induced transcriptional adaptation. These findings highlight a broad range of sensory neuron identities that are present at rest and that adapt to the experience of the individual, thus providing a novel layer of complexity to sensory coding.


title: Fractional Laser Treatment Boosts Systemic Anti-Tumor Immunity in Combination with OX40 Agonist and PD-1 Blockade

abstract: While ablative fractional photothermolysis (aFP) with a 10,600 nm CO2 laser is employed for a wide variety of dermatologic conditions, its applications in oncology are relatively unexplored. Building off our previous work, we investigated the effect of unilateral aFP treatment in combination with anti-PD-1 blocking antibody and OX40 agonist on bilateral tumor growth and remission. A CT26 wild type (CT26WT) colon carcinoma cell line was established bilaterally on the hind flanks of a standardized mouse model and tumor characteristics were investigated on aFP treated and untreated sides. Remarkably, triple therapy with fractional CO2 laser in combination with anti-PD-1 antibodies and OX40 agonists resulted in significantly slower tumor growth and complete remissions on bilateral tumors. Flow cytometric analysis showed the triple treatments elicited an increase of granzyme B+ CD8+T cells due to synergistic effect of aFP treatment and the checkpoint molecules, including the induction of CD103+ CCR7+ dendritic cells (DCs) in aFP-treated tumor by aFP treatment, XCR1+ DCs in drainage lymph node by anti-PD-1 inhibitor and OX40+ Ki67+ CD8+ T cells in the lymph node by OX40 agonist. Triple therapy-mediated tumor regression and survival was abrogated upon CD8+ T cell depletion. Importantly, when two mismatched cancer cells were implanted into mice, the effect of the triple therapy on distant tumor was abrogated, showing antigen specificity of the T cell immunity induced by triple therapy. This study highlights the efficacy of aFP a novel adjuvant for current cancer immunotherapeutics.


title: The impact of murine cytomegalovirus (mCMV) route and age at infection upon virus spread, immune responses and the establishment of latency

abstract: Cytomegalovirus (CMV) is a ubiquitous human virus, which establishes a characteristic lifetime infection in its host. Murine CMV (mCMV) is a widely-used infection model that has been employed to investigate the nature and extent of CMVs contribution to inflammatory, immunological, and health disturbances in humans. In an effort to assess the role of route and age in modeling hCMV infection in mice, we have performed a comparative analysis of two common experimental modes of infection (intraperitoneal and intranasal) at two different clinically relevant ages (4 weeks, or prepubescent childhood equivalent, and 12 weeks, or young postpubescent adult). We found that while both routes of infection led to similar early viral loads, differential activation of several parameters of innate immune function were observed. In particular, younger, prepubescent mice exhibited the strongest NK activation in the blood in response to i.p. infection, with this trend holding true in NK cells expressing the mCMV-specific receptor Ly49H. Moreover, i.p. infected animals accumulated a larger amount of anti-mCMV IgG and experienced a greater expansion of both acute and latent phase CD8+ T cells. This was especially true for young postpubescent mice, further illustrating a distinction in the bloodborne immune response across not only infection routes, but also ages. These results may be important in the understanding of how a more physiologically applicable model of CMV influences immunity, inflammation, and health over the lifespan.


title: Mapping nationally and globally at-risk species to identify hotspots for (and gaps in) conservation

abstract: Protecting habitat of species-at-risk is critical to their recovery, but can be contentious. For example, protecting species that are locally imperilled but globally common (e.g. species that only occur in a jurisdiction at the edge of their geographic range) is often thought to distract from protecting globally-imperilled species. However, such perceived trade-offs are based on the assumption that threatened groups have little spatial overlap, which is rarely quantified. Here, we compile range maps of terrestrial species-at-risk in Canada to assess the geographic overlap of nationally and globally at-risk species with each other, among taxonomic groups, and with protected areas. While many nationally-at-risk taxa only occurred in Canada at their northern range edge (median=4% of range in Canada), nationally-at-risk species were not significantly more peripheral in Canada than globally-at-risk species. Further, 56% of hotspots of nationally-at-risk taxa were also hotspots of globally-at-risk taxa in Canada, undercutting the perceived trade-off in their protection. Hotspots of nationally-at-risk taxa also strongly overlapped with hotspots of individual taxonomic groups, though less so for mammals. While strong spatial overlap across threat levels and taxa should facilitate efficient habitat protection, <7% of the area in Canadas at-risk hotspots is protected, and more than 70% of nationally and globally-at-risk species in Canada have <10% of their Canadian range protected. Our results counter the perception that protecting nationally vs. globally at-risk species are at odds, and identify critical areas to target as Canada strives to increase its protected areas and promote species-at-risk recovery.

category: ecology


title: Bee communities in canola are affected by landscape context and farm management

abstract: Bees are key pollinators that promote greater yield and seed quality of oilseed crops such as canola. Canola acreage has increased over 1,000% in the past decade in the Pacific Northwest USA, providing a major pulse of sugar-rich nectar and pollen resources that may affect bee health and community structure. However, because canola does not require insect pollination for seed production, few studies have examined the biodiversity of pollinators taking advantage of these resources, or the floral traits of canola that affect pollinators across variable landscapes. Here, we conducted pollinator surveys at canola farms across the inland Northwest region of the USA. We surveyed bee biodiversity and abundance, and assessed how these metrics correlated with landscape context, canola production practices, and floral traits of various canola varieties. We found that bee communities differed between sites and across growing seasons, with sweat bees more abundant later in the season, and mining bees more abundant earlier in the season. We also found that bees were more abundant overall on farms with less floral nectar and with less developed landscape surrounding the sampling area. Bee diversity was greater in spring canola than winter canola, and floral traits were also correlated with differing bee community diversity. This research provides important information for canola growers and land managers and offers a framework for future research in pollinator management in the inland Northwest.


title: Hybphaser identifies hybrid evolution in Australian Thelypterid ferns

abstract: Hybridisation can lead to reproductive isolation and consequently speciation. It has been proposed to play an important role in fern evolution, but has been difficult to investigate. This study explores the utility of target sequence capture and reference guided read phasing to investigate the role of evolutionary reticulation in ferns using Australian Thelypteridaceae as a model. The bioinformatics workflow HybPhaser was used to assess divergence between alleles, phase sequence reads to references to construct accessions resembling parental haplotpes, and include them in phylogenetic and network analyses to detect hybrids and parentage. This approach identified two novel hybrid lineages in Thelypteridaceae, one occurring between two different genera (Abacopteris and Christella), and provided evidence that reticulation is likely to have played an important role in the diversification of Australian thelypterids. In addition, hybrid phasing successfully reduced conflicting data and improved overall resolution in the Thelypteridaceae phylogeny, highlighting the power of this approach for reconstructing evolutionary history in reticulated lineages.

category: evolutionary biology


title: Theoretical Analysis of Principal Components in an Umbrella Model of Intraspecific Evolution

abstract: Principal component analysis (PCA) is one of the most frequently-used approach to describe population structure from multilocus genotype data. Regarding geographic range expansions of modern humans, interpretations of PCA have, however, been questioned, as there is uncertainty about the wave-like patterns that have been observed in principal components. It has indeed been argued that wave-like patterns are mathematical artifacts that arise generally when PCA is applied to data in which genetic differentiation increases with geographic distance. Here, we present an alternative theory for the observation of wave-like patterns in PCA. We study a coalescent model - the umbrella model - for the diffusion of genetic variants. The model is based on genetic drift without any particular geographical structure. In the umbrella model, splits from an ancestral population occur almost continuously in time, giving birth to small daughter populations at a regular pace. Our results provide detailed mathematical descriptions of eigenvalues and eigenvectors for the PCA of sampled genomic sequences under the model. Removing variants uniquely represented in the sample, the PCA eigenvectors are defined as cosine functions of increasing periodicity, reproducing wave-like patterns observed in equilibrium isolation-by-distance models. Including rare variants in the analysis, the eigenvectors corresponding to the largest eigenvalues exhibit complex wave shapes. The accuracy of our predictions is further investigated with coalescent simulations. Our analysis supports the hypothesis that highly structured wave-like patterns could arise from genetic drift only, and may not always be artificial outcomes of spatially structured data. Genomic data related to the peopling of the Americas are reanalyzed in the light of our new theory.


title: Rapid multilocus adaptation of clonal cabbage leaf curl virus populations to Arabidopsis thaliana

abstract: Cabbage leaf curl virus (CabLCV) has a bipartite single-stranded DNA genome and infects the model plant Arabidopsis thaliana. CabLCV serves as a model for the genus Begomovirus, members of which cause tremendous crop losses worldwide. We have used CabLCV as a model for within-plant virus evolution by inoculating individual plants with infectious clones of either a wild-type or mutagenized version of the CabLCV genome. Consistent with previous reports, detrimental substitutions in the Replication-associated gene (Rep) were readily compensated for by direct reversion and/or alternative mutations. A surprising number of common mutations were detected elsewhere in both viral segments (DNA-A and DNA-B) indicating convergent evolution and suggesting that CabLCV may not be as well adapted to A. thaliana as commonly presumed. Consistent with this idea, a spontaneous coat protein variant consistently rose to high allele frequency in susceptible accession Col-0, at a higher rate than in hypersusceptible accession Sei-0. Numerous high-frequency mutations were also detected in a candidate Rep binding site in DNA-B. Our results reinforce the fact that spontaneous mutation of this type of virus occurs rapidly and can change the majority consensus sequence of a within-plant virus population in weeks.