title: DHODH is an independent prognostic marker and potent therapeutic target in neuroblastoma

abstract: Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a pan-cancer, multi-omic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. By combining metabolomics, CRISPR screen and gene expression data from more than 700 solid tumor cell lines, we identified DHODH, a critical enzyme in pyrimidine synthesis, as a potential novel treatment target in neuroblastoma. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of high-risk neuroblastoma tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In neuroblastoma cell lines, DHODH gene dependency was found to correlate with MYCN dependency, rendering these cell lines highly sensitive to DHODH inhibition in vitro. In xenograft and transgenic neuroblastoma mouse models, tumor growth was dramatically reduced, and survival extended following treatment with the DHODH inhibitor brequinar. A combination of brequinar and temozolomide cured the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy with curative potential. Overall, DHODH inhibition combined with temozolomde has clear therapeutic potential in neuroblastoma and we propose this combination as a candidate for clinical testing.


title: Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with ACE2 interface, which gives 2G1 ability to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar IC50 in vitro. In SARS-CoV-2 and Beta- and Delta-variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 could be potentially capable of dealing with emerging SARS-CoV-2 variants in future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

category: cell biology


title: Screening key genes and signaling pathways in COVID-19 infection and its associated complications by integrated bioinformatics analysis

abstract: Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2)/ coronavirus disease 2019 (COVID-19) infection is the leading cause of respiratory tract infection associated mortality worldwide. The aim of the current investigation was to identify the differentially expressed genes (DEGs) and enriched pathways in COVID-19 infection and its associated complications by bioinformatics analysis, and to provide potential targets for diagnosis and treatment. Valid next-generation sequencing (NGS) data of 93 COVID 19 samples and 100 non COVID 19 samples (GSE156063) were obtained from the Gene Expression Omnibus database. Gene ontology (GO) and REACTOME pathway enrichment analysis was conducted to identify the biological role of DEGs. In addition, a protein-protein interaction network, modules, miRNA-hub gene regulatory network, TF-hub gene regulatory network and receiver operating characteristic curve (ROC) analysis were used to identify the key genes. A total of 738 DEGs were identified, including 415 up regulated genes and 323 down regulated genes. Most of the DEGs were significantly enriched in immune system process, cell communication, immune system and signaling by NTRK1 (TRKA). Through PPI, modules, miRNA-hub gene regulatory network, TF-hub gene regulatory network analysis, ESR1, UBD, FYN, STAT1, ISG15, EGR1, ARRB2, UBE2D1, PRKDC and FOS were selected as hub genes, which were expressed in COVID-19 samples relative to those in non COVID-19 samples, respectively. Among them, ESR1, UBD, FYN, STAT1, ISG15, EGR1, ARRB2, UBE2D1, PRKDC and FOS were suggested to be diagonstic factors for COVID-19. The findings from this bioinformatics analysis study identified molecular mechanisms and the key hub genes that might contribute to COVID-19 infection and its associated complications.

category: bioinformatics


title: Control of subunit stoichiometry in single-chain MspA nanopores

abstract:
Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

category: biophysics


title: LIGHTHOUSE illuminates therapeutics for a variety of diseases including COVID-19

abstract: One of the bottlenecks in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs for given therapeutic targets. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified novel therapeutics for cancer, lifestyle-related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. We envision that LIGHTHOUSE will bring about a paradigm shift in translational medicine, providing a bridge from bench side to bedside.

category: systems biology


title: Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and thrombosis

abstract: SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed 1. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11-/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out (Gsdmd-/-) mice. GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11-/- and Gsdmd-/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11-/- mice. We confirmed that protein levels of inflammatory mediators IL-1{beta}, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

category: immunology


title: LRRC15 mediates an accessory interaction with the SARS-CoV-2 spike protein

abstract:
The interactions between severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to COVID-19. The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary ACE2 receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was restricted to SARS-CoV-2, suggesting LRRC15 represents a novel class of spike binding interaction. We localized the interaction to the C-terminus of the S1 domain, and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19.

In briefWe present evidence from genome-wide screening that the spike protein of SARS-CoV-2 interacts with human cells expressing LRRC15. The interaction is distinct from previously known classes of spike attachment factors, and appears to have emerged recently within the coronavirus family. Although not sufficient for cell invasion, this interaction can modulate viral infection. Our data point to an unappreciated host factor for SARS-CoV-2, with potential relevance to COVID-19.

Highlights- Two systematic cell-based screens for SARS-CoV-2 spike protein binding identify LRRC15 as a human host factor
- Interaction with LRRC15 is reproducible in different human cell lines and independent of known glycan or ACE2 binding pathways
- The C-terminal S1 domain of SARS-CoV-2 spike binds LRRC15 with sub-micromolar affinity, while related coronavirus spikes do not
- LRRC15 is expressed in tissues with high ACE2 levels and may modulate infection

category: biochemistry


title: Protein Vaccine Induces a Durable, More Broadly Neutralizing Antibody Response in Macaques than Natural Infection with SARS-CoV-2 P.1

abstract:
FDA-approved and Emergency Use Authorized (EUA) vaccines using new mRNA and viral-vector technology are highly effective in preventing moderate to severe disease, however, information on their long-term efficacy and protective breadth against SARS-CoV-2 Variants of Concern (VOCs) is currently scarce. Here we describe the durability and broad-spectrum VOC immunity of a prefusion-stabilized spike (S) protein adjuvanted with liquid or lyophilized CoVaccine HT in cynomolgus macaques. This recombinant subunit vaccine is highly immunogenic and induces robust spike-specific and broadly neutralizing antibody responses effective against circulating VOCs (B.1.351 [Beta], P.1 [Gamma], B.1.617 [Delta]) for at least 3 months after the final boost. Protective efficacy and post-exposure immunity were evaluated using a heterologous P.1 challenge nearly 3 months after the last immunization. Our results indicate that while immunization with both high and low S doses shorten and reduce viral loads in the upper and lower respiratory tract, a higher antigen dose is required to provide durable protection against disease as vaccine immunity wanes. Histologically, P.1 infection causes similar COVID-19-like lung pathology as seen with early pandemic isolates. Post-challenge IgG concentrations were restored to peak immunity levels and vaccine-matched and cross-variant neutralizing antibodies were significantly elevated in immunized macaques indicating an efficient anamnestic response. Only low levels of P.1-specific neutralizing antibodies with limited breadth were observed in control (non-vaccinated but challenged) macaques suggesting that natural infection may not prevent reinfection by other VOCs. Overall, these results demonstrate that a properly dosed and adjuvanted recombinant subunit vaccine can provide long-lasting and protective immunity against circulating VOCs.

One Sentence SummaryA recombinant subunit protein formulated with CoVaccine HT adjuvant induces superior immunity than natural infection and reduces viral load while protecting cynomolgus macaques from COVID-19-like disease caused by late SARS-CoV-2 P.1 (Gamma) challenge.


title: Rapid in vivo development of resistance to daptomycin in vancomycin-resistant Enterococcus faecium due to genomic rearrangements

abstract:
BackgroundDaptomycin is a cyclic lipopeptide used in the treatment of vancomycin-resistant Enterococcus faecium (VREfm). However, the development of daptomycin-resistant VREfm challenges the treatment of nosocomial VREfm infections. Resistance mechanisms of daptomycin are not fully understood. Here we analysed the genomic changes leading to a daptomycin-susceptible VREfm isolate becoming resistant after 40 days of daptomycin and linezolid combination therapy.

MethodsThe two isogenic VREfm isolates (daptomycin-susceptible and daptomycin-resistant) were analysed using whole genome sequencing with Illumina and Nanopore.

ResultsWhole genome comparative analysis identified the loss of a 46.5 kb fragment and duplication of a 29.7 kb fragment in the daptomycin-resistant isolate, with many implicated genes involved in cell wall synthesis. Two plasmids of the daptomycin-susceptible isolate were also found integrated in the chromosome of the resistant isolate. One nonsynonymous SNP in the rpoC gene was identified in the daptomycin-resistant isolate.

ConclusionsDaptomycin resistance developed through chromosomal rearrangements leading to altered cell wall structure. Such novel types of resistance mechanisms can only be identified by comparing closed genomes of isogenic isolates.

category: microbiology


title: The chromosomal distribution of sex-biased microRNAs in Drosophila is non-adaptive

abstract: Genes are often differentially expressed between males and females. In Drosophila melanogaster, the analysis of sex-biased microRNAs (short non-coding regulatory molecules) has revealed striking differences with protein-coding genes. Mainly, the X chromosome is enriched in male-biased microRNA genes, although it is depleted of male-biased protein-coding genes. The paucity of male-biased genes in the X chromosome is generally explained by an evolutionary process called demasculinization. I suggest that the excess of male-biased microRNAs in the X chromosome is due to high-rates of de novo emergence of microRNAs (mostly in other neighboring microRNAs), a tendency of novel microRNAs in the X chromosome to be expressed in testis, and to a lack of a demasculinization process. To test this hypothesis I analysed the expression profile of microRNAs in males, females and gonads in D. pseudoobscura, in which an autosome translocated into the X chromosome effectively becoming part of a sex chromosome (neo-X). I found that the pattern of sex-biased expression is generally conserved between D. melanogaster and D. pseudoobscura. Also, orthologous microRNAs in both species conserve their chromosomal location, indicating that there is no evidence of demasculinization or other inter-chromosomal movement of microRNAs. D. pseudoobscura-specific microRNAs in the neo-X chromosome tend to be male-biased and particularly expressed in testis. In summary, the apparent paradox resulting from male-biased protein-coding genes depleted in the X chromosome and an enrichment in male-biased microRNAs is consistent with different evolutionary dynamics between coding genes and short RNAs.

category: evolutionary biology


title: The germline-specific region of the sea lamprey genome plays a key role in spermatogenesis

abstract: The sea lamprey genome undergoes programmed genome rearrangement (PGR) in which [~]20% is jettisoned from somatic cells soon after fertilization. Although the role of PGR in embryonic development has been studied, the role of the germline-specific region (GSR) in gonad development is unknown. We analysed RNA-sequence data from 28 sea lamprey gonads sampled across life-history stages, generated a genome-guided de novo superTransciptome with annotations, and identified genes in the GSR. We found that the 638 genes in the GSR are enriched for reproductive processes, exhibit 36x greater odds of being expressed in testes than ovaries, show little evidence of conserved synteny with other chordates, and most have putative paralogues in the GSR and/or somatic genomes. Further, several of these genes play known roles in sex determination and differentiation in other vertebrates. We conclude that the GSR of sea lamprey plays an important role in testicular differentiation and potentially sex determination.

category: genomics


title: Leafy and Weedy Seadragon Genomes Connect Genic and Repetitive DNA Features to the Extravagant Biology of Syngnathid Fishes

abstract: Seadragons are a remarkable lineage of teleost fishes, and they are members of the family Syngnathidae renowned for having evolved male pregnancy. Comprising three known species, seadragons are widely recognized and admired for their fantastical body forms and coloration, and their specific habitat requirements have made them flagship representatives for marine conservation and natural history interests. Until recently, a gap has been the lack of significant genomic resources for seadragons. We have produced gene-annotated, chromosome-scale genome models for the leafy and weedy seadragon to advance investigations into evolutionary innovation and elaboration of morphological traits in seadragons as well as their pipefish and seahorse relatives. We identified several interesting features specific to seadragon genomes, including divergent non-coding regions near a developmental gene important for integumentary outgrowth, a high genome-wide density of repetitive DNA, and recent expansions of transposable elements and a vesicular trafficking gene family. Surprisingly, comparative analyses leveraging the seadragon genomes and additional syngnathid and outgroup genomes revealed striking, syngnathid-specific losses in the family of fibroblast growth factors (FGFs), which likely involve re-organization of highly conserved gene regulatory networks in ways that have not previously been documented in natural populations. The resources presented here serve as important tools for future evolutionary studies of developmental processes in syngnathids and will be a key resource for conservation studies of the extravagant seadragons and their relatives.


title: Unsupervised deep learning reveals a stimulative effect of dopamine D3 receptor agonists on zebrafish sociality

abstract: It has been a major challenge to systematically evaluate and compare how pharmacological perturbations influence social behavioral outcomes. Although some pharmacological agents are known to alter social behavior, precise description and quantification of such effects have proven difficult. The complexity of brain functions regulating sociality makes it challenging to predict drug effects on social behavior without testing in live animals, and most existing behavioral assays are low-throughput and provide only unidimensional readouts of social function. To achieve richer characterization of drug effects on sociality, we developed a scalable social behavioral assay for zebrafish named ZeChat based on unsupervised deep learning. High-dimensional and dynamic social behavioral phenotypes are automatically classified using this method. By screening a neuroactive compound library, we found that different classes of chemicals evoke distinct patterns of social behavioral fingerprints. By examining these patterns, we discovered that dopamine D3 agonists possess a social stimulative effect on zebrafish. The D3 agonists pramipexole, piribedil, and 7-hydroxy-DPAT-HBr rescued social deficits in a valproic acid-induced zebrafish autism model. The ZeChat platform provides a promising approach for dissecting the pharmacology of social behavior and discovering novel social-modulatory compounds.

category: animal behavior and cognition


title: Social behavioral profiling by unsupervised deep learning reveals a stimulative effect of dopamine D3 agonists on zebrafish sociality


title: Identification of Bacteriophages Using Deep Representation Model with Pre-training

abstract:
Bacteriophages/Phages are viruses that infect and replicate within bacteria and archaea. Antibiotic resistance is one of the biggest threats to global health. The therapeutic use of bacteriophages provides another potential solution for solving antibiotic resistance. To develop phage therapies, the identification of phages from metagenome sequences is the fundamental step. Currently, several methods have been developed for identifying phages. These methods can be categorized into two types: database-based methods and alignment-free methods. The database-based approach, such as VIBRANT, utilizes existing databases and compares sequence similarity between candidates and those in the databases. The alignment-free method, such as Seeker and DeepVirFinder, uses deep learning models to directly predict phages based on nucleotide sequences. Both approaches have their advantages and disadvantages.

In this work, we propose using a deep representation learning model with pre-training to integrate the database-based and non-alignment-based methods (we call it INHERIT). The pre-training is used as an alternative way for acquiring knowledge representations from existing databases, while the BERT-style deep learning framework retains the advantage of alignment-free methods. We compared the proposed method with VIBRANT and Seeker on a third-party benchmark dataset. Our experiments show that INHERIT achieves better performance than the database-based approach and the alignment-free method, with the best F1-score of 0.9868. Meanwhile, we demonstrated that using pre-trained models helps to improve the non-alignment deep learning model further.


title: Bakdrive: Identifying the Minimum Set of Bacterial Driver Species across Multiple Microbial Communities

abstract: Interactions among microbes within microbial communities have been shown to play crucial roles in human health. In spite of recent progress, low-level knowledge of bacteria driving microbial interactions within microbiomes remains unknown, limiting our ability to fully understand and control microbial communities. In this study, we present a novel approach for identifying driver species within microbiomes. Bakdrive infers ecological networks of given metagenomic sequencing samples and identifies minimum sets of driver species using control theory. Bakdrive has three key innovations in this space: (i) it leverages inherent information from metagenomic sequencing samples to identify driver species, (ii) it explicitly takes host-specific variation into consideration, and (iii) it does not require a known ecological network. In extensive simulated data, we demonstrate identifying driver species identified from healthy donor samples and introducing them to the disease samples, we can restore the gut microbiome in recurrent Clostridioides difficile infection patients to a healthy state. We also applied Bakdrive to two real datasets, rCDI and Crohns disease patients, uncovering driver species consistent with previous work. In summary, Bakdrive provides a novel approach for teasing apart microbial interactions. Bakdrive is open-source and available at https://gitlab.com/treangenlab/bakdrive


title: Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants

abstract:
Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor belong to any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for such mutations. In this study, we present an analysis of 144 human enzymes containing 591 pathogenic missense variants, in which allosteric dynamic coupling of mutated positions with known active sites provides insights into a primary biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of {beta}-Glucocerebrosidase (GCase), which contains 94 Gaucher disease-associated missense variants located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling due to the presence of missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.

Author SummaryGenetic diseases occur when mutations to a particular gene cause a gain/loss in function of the related protein. Although several methods based on conservation and protein biochemistry exist to predict which genetic mutations may impact function, many disease causing changes remain unexplained by these metrics. In this study, we propose an explanation for these genetic changes may cause disease. In order to function, important regions of a protein must be able to exhibit collective motion. Through computer simulations, we observed that changing even a single amino acid within a protein can change the protein motion. Notably, disease causing genetic changes tend to alter the motion of regions which are critically important to protein function, even the mutations are far from these critical regions. In addition, we examined the degree that two amino acids within a protein may "couple" to one another, meaning the degree to which motion in one amino acid will affect the other. We found that amino acids which are highly coupled to the active site of a protein are more likely to result in disease if mutated, thereby offering a new tool for predicting genetic disease which incorporates internal protein dynamics.


title: Myofibril orientation as a metric for characterizing heart disease

abstract:
Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been due to a paucity of methods for objective and quantitative analysis. Here we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority as compared to conventional histological assessments. Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle {sigma}). At a sarcomere length (SL) of 1.9 m, the angle {sigma} was 0.23{+/-}0.01 rad, decreased to 0.19{+/-}0.01 rad at a SL of 2.1 m, and further decreased to 0.15{+/-}0.01 rad at a SL of 2.3 m (p<0.0001). Angle {sigma} was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy (HCM) model, porcine myocardium (0.24{+/-}0.01 rad) compared to WT myocardium (0.14{+/-}0.005 rad, p<0.0001) as well as in human heart failure tissue (0.19{+/-}0.006 rad) when compared to non-failing samples (0.17{+/-}0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared to healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological approaches that assess myocyte orientation and only indirectly evaluate myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen human myocardium provides a new tool for investigating structural anomalies associated with cardiac diseases.

Statement of SignificanceWe introduce a precise and quantitative approach to directly measure myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions as a novel tool for phenotypically characterizing striated muscle systems. We use this technique to demonstrate that myocardium from disease model organisms and failing human myocardium suffers from greater myofibrillar disorientation compared to healthy controls. We also demonstrate that excellent diffraction patterns can be obtained from frozen and thawed human myocardium. Given the ready availability of frozen human heart tissue in tissue banks, this capability opens up a large space of potential experiments relating sarcomere structure to dysfunction in cardiac disorders.


title: Single-cell RNA Sequencing Reveals Immunosuppressive Myeloid Cell Diversity and Restricted Cytotoxic Effector Cell Trafficking and Activation During Malignant Progression in Glioma

abstract: Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a marker of tumor associated macrophages. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II gliomas. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

category: cancer biology


title: Cancer missense mutations in the BRC repeats of BRCA2 protein disrupt RAD51 binding and activity leading to chemotherapeutic sensitivity

abstract:
Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic, however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

Graphical Abstract

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title: BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair


title: Attachment strength of seed mucilage prevents seed dislodgement in high surface flow: a mechanistic investigation

abstract:
O_LIBackground and Aims: Seed mucilage is a common and highly diverse trait shared among thousands of angiosperm species. While long recognized that mucilage allows seeds to anchor to substrates (antitelechory), resisting abiotic and biotic dislodgement, we still lack a mechanistic understanding of this process.
C_LIO_LIMethods: We propose a mechanistic model of how mucilage affects substrate anchorage and fluid resistance, ultimately contributing to dislodgement resistance. To test this model, we subjected mucilaginous seeds of 52 species, varying in eight measured seed traits, to seven days of continuous water flow at a range of dislodgement potentials.
C_LIO_LIKey Results: Supporting our model, mucilage mass increased force necessary to dislodge both dry and wet seeds; our measurement of the dislodgement force of dry mucilage explained time to dislodgement well. The effect size was remarkably large; increasing the standardized mucilage mass by one standard deviation resulted in a 280-fold increase in the time to dislodgement. Fluid resistance was largely dependent on speed of water flow and the seeds modified drag coefficient, but not seed traits. Neither mucilage expansion speed nor mucilage decay rate explained dislodgement potential well.
C_LIO_LIConclusions: Our results suggest that the degree of anchorage to substrate, measured with a simple dislodgement force assay, is highly predictive of mucilaginous seed retention in highly erosive environments. In contrast, we found that other seed and mucilage traits are of lesser importance to anchorage.
C_LI

category: plant biology


title: Anchorage by seed mucilage prevents seed dislodgement in high surface flow: a mechanistic investigation


title: Instant Taq: Rapid Autoinducible Expression and Chromatography-free Purification of Taq polymerase.

abstract:
DNA modifying enzymes are ubiquitous reagents in synthetic biology. Producing these enzymes often requires large culture volumes, purified nucleases and chromatographic separations to make enzymes of necessary quality. We sought to leverage synthetic biology tools to develop engineered strains allowing for not only the production but rapid purification of these reagents. Toward this goal, we report an E. coli strain enabling the rapid production and purification of Taq polymerase. The method relies on 1) autoinducible expression achieving high protein titers, 2) autolysis and auto DNA/RNA hydrolysis via lysozyme and a mutant benzonase, and 3) heat denaturation under reducing conditions to precipitate contaminating proteins including the mutant benzonase. Taq polymerase is obtained at high purities (>95% pure by SDS-PAGE) and is readily usable in standard reactions. The method takes less than 1 hour of hands-on time, does not require special equipment, expensive reagents or affinity purification. We expect this simple methodology and approach will improve access not only to Taq polymerase but to numerous additional commonly utilized reagent proteins.

HighlightsO_LIProtein titers ~ 1g/L achieved in 20 mL shakeflasks.
C_LIO_LI4 mg of purified Taq (corresponding to 5,000 Units, or 4,000 PCR reactions) per 20 mL shake flask.
C_LIO_LIInstant Taq is equivalent to commercial preparations in routine PCR
C_LI

category: synthetic biology


title: 'Wanting' versus 'Needing' related value: an fMRI meta-analysis

abstract: Consumption and its excesses are sometimes explained by imbalance of need or lack of control over  wanting.  Wanting assigns value to cues that predict rewards, whereas  needing assigns value to biologically significant stimuli that one is deprived of. Here we aimed at studying how the brain activation patterns related to value of  wanted stimuli differs from that of  needed stimuli using ALE neuroimaging meta-analysis approaches. We used the perception of a cue predicting a reward for  wanting related value and the perception of food stimuli in a hungry state as a model for  needing related value. We carried out separate, contrasts, and conjunction meta-analyses to identify differences and similarities between  wanting and  needing values. Our overall results for  wanting related value show consistent activation of the ventral tegmental area, striatum and pallidum, regions that both activate behaviour and direct choice; while for  needing related value we found an overall consistent activation of the middle insula and to some extent the caudal-ventral putamen, regions that only direct choice. Our study suggests that wanting has more control on consumption, and a needed stimuli must become wanted in order to be pursued.

category: neuroscience


title: Express arm Responses Appear Bilaterally on Upper-limb Muscles in Arm Choice Reaching Task

abstract:
When required, humans can generate very short latency reaches towards visual targets, like catching a falling cellphone. During such rapid reaches, express arm responses are the first wave of upper limb muscle recruitment, occurring ~80-100 ms after target appearance. There is accumulating evidence that express arm responses arise from signaling along the tecto-reticulo-spinal tract, but the involvement of the reticulo-spinal tract has not been well-studied. Since the reticulospinal tract projects bilaterally, we studied whether express arm responses would be generated bilaterally. Human participants (n = 14; 7 female) performed visually guided reaches in a modified emerging target paradigm where either arm could intercept the target. We recorded electromyographic activity bilaterally from the pectoralis major muscle. Our analysis focused on target locations where participants reached with the right arm on some trials, and the left arm on others. In support of the involvement of the reticulospinal tract, express arm responses persisted bilaterally regardless of which arm reached to the target. The latency and magnitude of the express arm response did not depend on whether the arm was chosen to reach or not. However, on the reaching arm, the magnitude of the express arm response was correlated to the level of anticipatory activity. The bilateral generation of express arm responses supports the involvement of the reticulo-spinal tract. We surmise that the correlation between anticipatory activity and the magnitude of express arm responses on the reaching arm arises from convergence of cortically-derived signals with a parallel subcortical pathway mediating the express arm response.

New and NoteworthyExpress arm responses have been proposed to arise from the tecto-reticulo-spinal tract originating within the superior colliculus, but the involvement of the reticulo-spinal tract has not been well studied. Here, we show these responses appear bilaterally in a task where either arm can reach to a newly appearing stimulus. Our results suggest that the most rapid visuomotor transformations for reaching are performed by a subcortical pathway.


title: Express Arm Responses Appear Bilaterally on Upper-limb Muscles in An Arm Choice Reaching Task


title: Region-specific KCC2 rescue by rhIGF-1 and oxytocin in a mouse model of Rett syndrome

abstract: Rett syndrome (RTT) is characterized by dysfunction in neuronal excitation/inhibition (E/I) balance, potentially impacting seizure susceptibility via deficits in K+/Cl- cotransporter 2 (KCC2) function. Mice lacking the Methyl-CpG binding protein 2 (MeCP2) recapitulate many symptoms of RTT, and recombinant human insulin-like growth factor-1 (rhIGF-1) restores KCC2 expression and E/I balance in MeCP2 KO mice. However, clinical trial outcomes of rhIGF-1 in RTT have been variable, and increasing its therapeutic efficacy is highly desirable. To this end, the neuropeptide oxytocin (OXT) is promising, as it also critically modulates KCC2 function during early postnatal development. We measured basal KCC2 expression levels in MeCP2 KO mice and identified three key frontal brain regions showing KCC2 alterations in young adult mice but not in postnatal P10 animals. We thus hypothesized that deficits in an IGF-1/OXT signaling crosstalk modulating KCC2 may occur in RTT during postnatal development. Consistently, we detected alterations of IGF-1 receptor (IGF-1R) and OXT receptor (OXTR) levels in those brain areas. rhIGF-1 and OXT treatments in KO mice rescued KCC2 expression in a region-specific and complementary manner. These results suggest that region-selective combinatorial pharmacotherapeutic strategies could be the most effective at normalizing E/I balance in key brain regions subtending the RTT pathophysiology.


title: Kinetic and thermodynamic modeling of a voltage-gated sodium channel

abstract: Like all biological and chemical reactions, ion channel kinetics are highly sensitive to changes in temperature. Therefore, it is prudent to investigate channel dynamics at physiological temperatures. However, most ion channel investigations are performed at room temperature due to practical considerations, such as recording stability and technical limitations. This problem is especially severe for the fast voltage-gated sodium channel, whose activation kinetics are faster than the time constant of the standard patch-clamp amplifier at physiological temperatures. Thus, biologically detailed simulations of the action potential generation evenly scale the kinetic models of voltage-gated channels acquired at room temperature. To quantitatively study voltage-gated sodium channels temperature sensitivity, we recorded sodium currents from nucleated patches extracted from the rats layer five neocortical pyramidal neurons at several temperatures from 13.5 to 30oC. We use these recordings to model the kinetics of the voltage-gated sodium channel as a function of temperature. We show that the temperature dependence of activation differs from that of inactivation. Furthermore, we show that the sustained current has a different temperature dependence than the fast current. Our kinetic and thermodynamic analysis of the current provided a numerical model spanning the entire temperature range. This model reproduced vital features of channel activation and inactivation. Furthermore, the model also reproduced action potential dependence on temperature. Thus, we provide an essential building block for the generation of biologically detailed models of cortical neurons.


title: Direct reprogramming of the intestinal epithelium by parasitic helminths subverts type 2 immunity

abstract: Enteric helminths form intimate physical connections with the intestinal epithelium, yet their ability to directly alter epithelial stem cell fate has not been resolved. Here we demonstrate that infection of mice with the symbiotic parasite Heligmosomoides polygyrus bakeri (Hbp), reprograms the intestinal epithelium into a fetal-like state marked by the emergence of Clusterin-expressing revival stem cells (revSCs). Organoid-based studies using parasite-derived excretory/secretory products reveal that Hpb-mediated revSC generation occurs independent of host-derived immune signals and inhibits type 2 cytokine-driven differentiation of secretory epithelial lineages that promote their expulsion. Reciprocally, type 2 cytokine signals limit revSC differentiation and, consequently, Hpb fitness indicating that helminths compete with their host for control of the intestinal stem cell niche to promote continuation of their life cycle.


title: Genetic interplay between type II topoisomerase enzymes and chromosomal ccdAB toxin-antitoxin in E. coli

abstract: Fluoroquinolones are one of the most widely used class of antibiotics. They target two type II topoisomerase enzymes: gyrase and topoisomerase IV. Resistance to these drugs, which is largely caused by mutations in their target enzymes, is on the rise and becoming a serious public health risk. In this work, we analyze the sequences of 352 extraintestinal E. coli clinical isolates to gain insights into the selective pressures shaping the type II topoisomerase mutation landscape in E. coli. We identify both Quinolone Resistance-Determining Region (QRDR) and non-QRDR mutations, outline their mutation trajectories, and show that they are likely driven by different selective pressures. We confirm that ciprofloxacin resistance is specifically and strongly associated with QRDR mutations. By contrast, non-QRDR mutations are associated with the presence of the chromosomal version of ccdAB, a toxin-antitoxin operon, where the toxin CcdB is known to target gyrase. We also find that ccdAB and the evolution of QRDR mutation trajectories are partially incompatible. Finally, we identify partial deletions in CcdB and additional mutations that likely facilitate the compatibility between the presence of the ccdAB operon and QRDR mutations. These "permissive" mutations are all found in ParC (a topoisomerase IV subunit). This, and the fact that CcdB-selected mutations frequently map to topoisomerase IV, strongly suggests that this enzyme (in addition to gyrase) is likely a target for the toxin CcdB in E. coli, although an indirect effect on global supercoiling cannot be excluded. This work opens the door for the use of the presence of ccdB and of the proposed permissive mutations in the genome as genetic markers to assess the risk of quinolone resistance evolution and implies that certain strains may be genetically more refractory to evolving quinolone resistance through mutations in target enzymes.


title: FSTruct: An Fst-based tool for measuring ancestry variation in inference of population structure

abstract: In model-based inference of population structure from individual-level genetic data, individuals are assigned membership coefficients in a series of statistical clusters generated by clustering algorithms. Distinct patterns of variability in membership coefficients can be produced for different groups of individuals, for example, representing different predefined populations, sampling sites, or time periods. Such variability can be difficult to capture in a single numerical value; membership coefficient vectors are multivariate and potentially incommensurable across groups, as the number of clusters over which individuals are distributed can vary among groups of interest. Further, two groups might share few clusters in common, so that membership coefficient vectors are concentrated on different clusters. We introduce a method for measuring the variability of membership coefficients of individuals in a predefined group, making use of an analogy between variability across individuals in membership coefficient vectors and variation across populations in allele frequency vectors. We show that in a model in which membership coefficient vectors in a population follow a Dirichlet distribution, the measure increases linearly with a parameter describing the variance of a specified component of the membership vector. We apply the approach, which makes use of a normalized FST statistic, to data on inferred population structure in three example scenarios. We also introduce a bootstrap test for equivalence of two or more groups in their level of membership coefficient variability. Our methods are implemented in the R package FSTruct.


title: Means, motive, and opportunity for biological invasions: genetic introgression in a fungal pathogen

abstract: Invasions by fungal plant pathogens pose a significant threat to the health of agriculture ecosystems. Despite limited standing genetic variation, many invasive fungal species can adapt and spread rapidly, resulting in significant losses in crop yields. Here, we report on the population genomics of Colletotrichum truncatum, a polyphagous pathogen that can infect more than 460 plant species, and an invasive pathogen on soybean in Brazil. We study the whole-genome sequences of 18 isolates representing 10 fields from two major regions of soybean production. We show that Brazilian C. truncatum is subdivided into three phylogenetically distinct lineages that exchange genetic variation through hybridization. Introgression affects 2 to 30% of the nucleotides of genomes and varies widely between the lineages. We find that introgressed regions comprise secreted protein-encoding genes, suggesting possible co-evolutionary targets for selection in those regions. We highlight the inherent vulnerability of genetically uniform crops in the agro-ecological environment, particularly when faced with pathogens that can take full advantage of the opportunities offered by an increasingly globalized world. Finally, we discuss "The Means, Motive, and Opportunity" of fungal pathogens and how they can become invasive species of crops. We call for more population genomic studies because such analyses can help identify geographic areas and pathogens that pose a risk, thereby helping to inform control strategies to better protect crops in the future.


title: Transcriptome-wide association and prediction for carotenoids and tocochromanols in fresh sweet corn kernels

abstract:
Sweet corn is consistently one of the most highly consumed vegetables in the U.S., providing a valuable opportunity to increase nutrient intake through biofortification. Significant variation for carotenoid (provitamin A, lutein, zeaxanthin) and tocochromanol (vitamin E, antioxidants) levels is present in temperate sweet corn germplasm, yet previous genome-wide association studies (GWAS) of these traits have been limited by low statistical power and mapping resolution. Here, we employed a high-quality transcriptomic dataset collected from fresh sweet corn kernels to conduct transcriptome-wide association studies (TWAS) and transcriptome prediction studies for 39 carotenoid and tocochromanol traits. In agreement with previous GWAS findings, TWAS detected significant associations for four causal genes, {beta}-carotene hydroxylase (crtRB1), lycopene epsilon cyclase (lcyE),{gamma} -tocopherol methyltransferase (vte4), and homogentisate geranylgeranyltransferase (hggt1) on a transcriptome-wide level. Pathway-level analysis revealed additional associations for deoxy-xylulose synthase2 (dxs2), diphosphocytidyl methyl erythritol synthase2 (dmes2), cytidine methyl kinase1 (cmk1), and geranylgeranyl hydrogenase1 (ggh1), of which, dmes2, cmk1, and ggh1 have not previously been identified through maize association studies. Evaluation of prediction models incorporating genome-wide markers and transcriptome-wide abundances revealed a trait-dependent benefit to the inclusion of both genomic and transcriptomic data over solely genomic data, but both transcriptome- and genome-wide datasets outperformed a priori candidate gene-targeted prediction models for most traits. Altogether, this study represents an important step towards understanding the role of regulatory variation in the accumulation of vitamins in fresh sweet corn kernels.

Core IdeasO_LITranscriptomic data aid the study of vitamin levels in fresh sweet corn kernels.
C_LIO_LIcrtRB1, lcyE, dxs2, dmes2, and cmk1 were associated with carotenoid traits.
C_LIO_LIvte4, hggt1, and ggh1 were associated with tocochromanol traits.
C_LIO_LITranscriptomic data boosted predictive ability over genomic data alone for some traits.
C_LIO_LIJoint transcriptome- and genome-wide models achieved the highest predictive abilities.
C_LI


title: Glycerol-driven Denitratation: Process Kinetics, Microbial Ecology, and Operational Controls

abstract: Denitratation, the selective reduction of nitrate to nitrite, is a novel process when coupled with anaerobic ammonium oxidation (anammox) could achieve resource-efficient biological nitrogen removal of ammonium- and nitrate-laden waste streams. Using a fundamentally-based, first principles approach, this study optimized a stoichiometrically-limited, glycerol-driven denitratation process and characterized mechanisms supporting nitrite accumulation with results that aligned with expectations. Glycerol supported selective nitrate reduction to nitrite and near-complete nitrate conversion, indicating its viability in a denitratation system. Glycerol-supported specific rates of nitrate reduction (135.3 mg-N/g-VSS/h) were at least one order of magnitude greater than specific rates of nitrite reduction (14.9 mg-N/g-VSS/h), potentially resulting in transient nitrite accumulation and indicating glycerols superiority over other organic carbon sources in denitratation systems. pH and ORP inflection points in nitrogen transformation assays corresponded to maximum nitrite accumulation, indicating operational setpoints to prevent further nitrite reduction. Denitratation conditions supported enrichment of Thauera sp. as the dominant genus. Stoichiometric limitation of influent organic carbon, coupled with differential nitrate and nitrite reduction kinetics, optimized operational controls, and a distinctively enriched microbial ecology, was identified as causal in glycerol-driven denitratation.

category: bioengineering


title: Elucidation of sub-cellular H2S metabolism in Solanum lycopersicum L. and its assessment under development and biotic stress

abstract: The signalling molecules serve as a fundamental requirement in plants and respond to various internal and external cues. Among several signalling molecules, the significance of gasotransmitters has been realized in several plant developmental and environmental constraints. The hydrogen sulfide (H2S) is a novel signalling molecule in higher plants and is involved in several physiological processes right from seed germination to flowering and fruit ripening. Moreover, H2S also assist plants in managing biotic and abiotic stresses, therefore serves as one of the imperative choice of chemical priming. Yet, the metabolism of H2S is not much explored and only appraisal study is made till date from Arabidopsis thaliana. Therefore, the present investigation explored the elucidation of H2S metabolism in crop plant Solanum lycopersicum L. Through in silico investigations the study demonstrated the participation of 29 proteins involved in H2S metabolism, which are mainly localized in cytosol, chloroplast, and mitochondria. Additionally, the relevant protein-protein interactomes were also inferred for sub-cellular compartments and expression data were explored under development and biotic stresses namely PAMPs treatment and bacterial infection. The information generated here will be of high relevance to better target the H2S metabolism to enhance the tomato prospects and also serve a preliminary investigation to be adopted in other agronomic important crops.


title: The wheat dioxygenase BX6 is involved in the formation of benzoxazinoids  in planta  and contributes to plant defense against insect herbivores

abstract: Benzoxazinoids are plant specialized metabolites with defense properties, highly abundant in wheat (Triticum), one of the worlds most important crops. The goal of our study was to characterize dioxygenase BX6 genes in tetraploid and hexaploid wheat genotypes and to elucidate their effects on defense against herbivores. Phylogenetic analysis revealed four BX6 genes in the hexaploid wheat T. aestivum, but only one ortholog was found in tetraploid (T. turgidum) wild emmer wheat and the cultivated durum wheat. Transcriptome sequencing of durum wheat plants damaged either by aphids or caterpillars revealed that several BX genes including TtBX6 were upregulated upon caterpillar feeding relative to undamaged control plants. A virus-induced gene silencing approach was used to reduce the expression of BX6 in T. aestivum plants and exhibited both reduced transcript levels and reduced accumulation of different benzoxazinoids. To elucidate the effect of BX6 on plant defense, bioassays with different herbivores feeding on BX6-silenced leaves were conducted. The results showed that plants with silenced BX6 were more susceptible to aphids and the two-spotted spider mite compared to controls. Overall, our study indicates that wheat BX6 is involved in the formation of benzoxazinoids in planta and contributes to plant resistance against insect herbivores.


title: Post-trauma behavioral phenotype predicts vulnerability to fear relapse after extinction

abstract: Current treatments for trauma-related disorders remain ineffective for many patients. Here, we modeled interindividual differences in post-therapy fear relapse with a novel ethologically relevant trauma recovery paradigm. After traumatic fear conditioning, male rats underwent fear extinction while foraging in a large enriched arena, permitting the expression of a wide spectrum of behaviors, assessed by an automated pipeline. This multidimensional behavioral assessment revealed that post-conditioning fear response profiles clustered into two groups, respectively characterized by active vs. passive fear responses. After trauma, some animals expressed fear by freezing, while others darted, as if fleeing from danger. Remarkably, belonging to the darters or freezers group predicted differential levels of vulnerability to fear relapse after extinction. Moreover, genome-wide transcriptional profiling revealed that these groups differentially regulated specific sets of genes, some of which have previously been implicated in anxiety and trauma-related disorders. Our results suggest that post-trauma behavioral phenotypes and the associated epigenetic landscapes can serve as markers of fear relapse susceptibility, and thus may be instrumental for future development of more effective treatments for psychiatric patients.


title: Tuning in scene-preferring cortex for mid-level visual features gives rise to selectivity across multiple levels of stimulus complexity

abstract:
The scene-preferring portion of the human ventral visual stream, known as the parahippocampal place area (PPA), responds to scenes and landmark objects, which tend to be large in real-world size, fixed in location, and inanimate. However, the PPA also exhibits preferences for low-level contour statistics, including rectilinearity and cardinal orientations, that are not directly predicted by theories of scene- and landmark-selectivity. It is unknown whether these divergent findings of both low- and high-level selectivity in the PPA can be explained by a unified computational theory. To address this issue, we fit feedforward computational models of visual feature coding to the image-evoked fMRI responses of the PPA, and we performed a series of high-throughput experiments on these models. Our findings show that feedforward models of the PPA exhibit emergent selectivity across multiple levels of complexity, giving rise to seemingly high-level preferences for scenes and for objects that are large, spatially fixed, and inanimate/manmade while simultaneously yielding low-level preferences for rectilinear shapes and cardinal orientations. These results reconcile disparate theories of PPA function in a unified model of feedforward feature coding, and they demonstrate how multifaceted selectivity profiles naturally emerge from the feedforward computations of visual cortex and the natural statistics of images.

SIGNIFICANCE STATEMENTVisual neuroscientists characterize cortical selectivity by identifying stimuli that drive regional responses. A perplexing finding is that many higher-order visual regions exhibit selectivity profiles spanning multiple levels of complexity: they respond to highly complex categories, such as scenes and landmarks, but also to surprisingly simplistic features, such as specific contour orientations. Using large-scale computational analyses and human brain imaging, we show how multifaceted selectivity in scene-preferring cortex can emerge from the feedforward, hierarchical coding of visual features. Our work reconciles seemingly divergent findings of selectivity in scene-preferring cortex and suggests that surprisingly simple feedforward feature representations may be central to the category-selective organization of the human visual system.


title: Emergent selectivity for scenes, object properties, and contour statistics in feedforward models of scene-preferring cortex


title: Auditory Hypersensitivity and Processing Deficits in a Rat Model of Fragile X Syndrome

abstract: Fragile X (FX) syndrome is one of the leading inherited causes of autism spectrum disorder (ASD). A majority of FX and ASD patients exhibit sensory hypersensitivity, including auditory hypersensitivity or hyperacusis, a condition in which everyday sounds are perceived as much louder than normal. Auditory processing deficits in FX and ASD also afford the opportunity to develop objective and quantifiable outcome measures that are likely to translate between humans and animal models due to the well-conserved nature of the auditory system and well-developed behavioral read-outs of sound perception. Therefore, in this study we characterized auditory hypersensitivity in a Fmr1 knockout (KO) transgenic rat model of FX using an operant conditioning task to assess sound detection thresholds and suprathreshold auditory reaction time-intensity (RT-I) functions, a reliable psychoacoustic measure of loudness growth, at a variety of stimulus frequencies, bandwidths and durations. Male Fmr1 KO and littermate WT rats both learned the task at the same rate and exhibited normal hearing thresholds. However, Fmr1 KO rats had faster auditory RTs over a broad range of intensities and steeper RT-I slopes than WT controls, perceptual evidence of excessive loudness growth in Fmr1 KO rats. Furthermore, we found that Fmr1 KO animals exhibited abnormal perceptual integration of sound duration and bandwidth, with diminished temporal but enhanced spectral integration of sound intensity. Because temporal and spectral integration of sound stimuli were altered in opposite directions in Fmr1 KO rats, this suggests that abnormal RTs in these animals are evidence of aberrant auditory processing rather than generalized hyperactivity or altered motor responses. Together, these results are indicative of fundamental changes to low-level auditory processing in Fmr1 KO animals. Finally, we demonstrated that antagonism of metabotropic glutamate receptor 5 (mGlu5) selectively and dose-dependently restored normal loudness growth in Fmr1 KO rats, suggesting a pharmacologic approach for alleviating sensory hypersensitivity associated with FX. This study leverages the tractable nature of the auditory system and the unique behavioral advantages of rats to provide important insights into the nature of a centrally important yet understudied aspect of FX and ASD.


title: Normative and mechanistic model of an adaptive circuit for efficient encoding and feature extraction

abstract:
One major question in neuroscience is how to relate connectomes to neural activity, circuit function, and learning. We offer an answer in the peripheral olfactory circuit of the Drosophila larva, composed of olfactory receptor neurons (ORNs) connected through feedback loops with interconnected inhibitory local neurons (LNs). We combine structural and activity data and, using a holistic normative framework based on similarity-matching, we propose a biologically plausible mechanistic model of the circuit. Our model predicts the ORN [->] LN synaptic weights found in the connectome and demonstrate that they reflect correlations in ORN activity patterns. Additionally, our model explains the relation between ORN [->] LN and LN - LN synaptic weight and the arising of different LN types. This global synaptic organization can autonomously arise through Hebbian plasticity, and thus allows the circuit to adapt to different environments in an unsupervised manner. Functionally, we propose LNs extract redundant input correlations and dampen them in ORNs, thus partially whitening and normalizing the stimulus representations in ORNs. Our work proposes a comprehensive framework to combine structure, activity, function, and learning, and uncovers a general and potent circuit motif that can learn and extract significant input features and render stimulus representations more efficient.

SignificanceThe brain represents information with patterns of neural activity. At the periphery, due to the properties of the external world and of encoding neurons, these patterns contain correlations, which are detrimental for stimulus discrimination. We study the peripheral olfactory neural circuit of the Drosophila larva, that preprocesses neural representations before relaying them to higher brain areas. A comprehensive understanding of this preprocessing is, however, lacking. Here, we propose a mechanistic and normative framework describing the function of the circuit and predict the circuits synaptic organization based on the circuits input neural activity. We show how the circuit can autonomously adapt to different environments, extracts stimulus features, and decorrelate and normalize input representations, which facilitates odor discrimination downstream.


title: Stochastic modeling uncovers a novel mechanism underlying the evolution of mutation rates in sexually reproducing populations

abstract: Natural selection is believed to universally work to lower mutation rates (MR) due to the negative impact of mutations on individual fitness. Mutator alleles have only been found to be co-selected by genetic linkage with adaptive alleles in prokaryotes. Sexual reproduction substantially reduces genetic linkage, allowing selection to effectively eradicate mutator alleles. The current understanding, therefore, is that in sexually reproducing populations selection always works to lower MR, limited by the effective population size that determines the overall selection efficiency. In the present paper, we apply a Monte Carlo model of a sexually reproducing population and demonstrate that selection acting on MR does not universally favor lower MR but depends on the mode of selection acting on adaptive phenotypic traits. We demonstrate a unique previously unreported co-selective process that can drive the evolution of higher MR in sexually reproducing populations. Our results show that MR evolution is significantly influenced by multigenic inheritance of both MR and adaptive traits that are under selection. Our results also show that, contrary to the generally accepted axiom, population size appears not to affect the strength of selection uniformly but likely forms an intra-population gradient that generates a "biased sampling" process that has an opposite effect on selection strength and thus modulates or even negates the effect of population size on MR evolution. Based on our results, we propose an expanded population genetics theory of the evolution of mutation rates in sexually reproducing organisms. Our results have potential implications for understanding processes underlying rapid adaptive change in speciation and related macroevolutionary patterns.


title: A neural network-based method for exhaustive cell label assignment using single cell RNA-seq data

abstract: The fast-advancing single cell RNA sequencing (scRNA-seq) technology enables researchers to study the transcriptome of heterogeneous tissues at a single cell level. The initial important step of analyzing scRNA-seq data is usually to accurately annotate cells. The traditional approach of annotating cell types based on unsupervised clustering and marker genes is time-consuming and laborious. Taking advantage of the numerous existing scRNA-seq databases, many supervised label assignment methods have been developed. One feature that many label assignment methods shares is to label cells with low confidence as "unassigned." These unassigned cells can be the result of assignment difficulties due to highly similar cell types or caused by the presence of unknown cell types. However, when unknown cell types are not expected, existing methods still label a considerable number of cells as unassigned, which is not desirable. In this work, we develop a neural network-based cell annotation method called NeuCA (Neural network-based Cell Annotation) for scRNA-seq data obtained from well-studied tissues. NeuCA can utilize the hierarchical structure information of the cell types to improve the annotation accuracy, which is especially helpful when data contain closely correlated cell types. We show that NeuCA can achieve more accurate cell annotation results compared with existing methods. Additionally, the applications on eight real datasets show that NeuCA has stable performance for intra- and inter-study annotation, as well as cross-condition annotation. NeuCA is freely available as an R/Bioconductor package at https://bioconductor.org/packages/NeuCA.


title: Cross-species cell-type assignment of single-cell RNA-seq by a heterogeneous graph neural network

abstract: Cross-species comparative analyses of single-cell RNA sequencing (scRNA-seq) data allow us to explore, at single-cell resolution, the origins of cellular diversity and the evolutionary mechanisms that shape cellular form and function. Here, we aimed to utilize a heterogeneous graph neural network to learn aligned and interpretable cell and gene embeddings for cross-species cell type assignment and gene module extraction (CAME) from scRNA-seq data. A systematic evaluation study on 649 pairs of cross-species datasets showed that CAME outperformed six benchmarking methods in terms of cell-type assignment and model robustness to insufficiency and inconsistency of sequencing depths. Comparative analyses of the major types of human and mouse brains by CAME revealed shared cell type-specific functions in homologous gene modules. Alignment of the trajectories of human and macaque spermatogenesis by CAME revealed conservative gene expression dynamics during spermatogenesis between humans and macaques. Owing to the utilization of non-one-to-one homologous gene mappings, CAME made a significant improvement on cell-type characterization cross zebrafish and other species. Overall, CAME can not only make an effective cross-species assignment of cell types on scRNA-seq data but also reveal evolutionary conservative and divergent features between species.


title: Summarizing internal dynamics boosts differential analysis and functional interpretation of super enhancers

abstract: Super enhancers (SEs) are broad enhancer domains usually containing multiple constituent enhancers that hold elevated activities in gene regulation. Disruption in one or more constituent enhancers causes aberrant SE activities that lead to gene dysregulation in diseases. To quantify SE aberrations, differential analysis is performed to compare SE activities between cell conditions. The state-of-art strategy in estimating differential SEs relies on overall activities and neglect the changes in length and structure of SEs. Here, we propose a novel computational method to identify differential SEs by weighting the combinatorial effects of constituent-enhancer activities and locations (i.e., internal dynamics). In addition to overall activity changes, our method identified four novel classes of differential SEs with distinct enhancer structural alterations. We demonstrate that these structure alterations hold distinct regulatory impact, such as regulating different number of genes and modulating gene expression with different strengths, highlighting the differentiated regulatory roles of these unexplored SE features. When compared to the existing method, our method showed improved identification of differential SEs that were linked to better discernment of cell-type-specific SE activity and functional interpretation. We implement an R package, DASE, to facilitate the use of our method.


title: Single-molecule analysis of specificity and multivalency in binding of short linear substrate motifs to the APC/C

abstract: Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.


title: Large-scale comparative small RNA analyses reveal genomic structural variants in driving expression dynamics and differential selection pressures on distinct small RNA classes during tomato domestication

abstract: Tomato has undergone extensive selections during domestication. Recent progress has shown that genomic structural variants (SVs) have contributed to gene expression dynamics during tomato domestication, resulting in changes of important traits. Here, through comprehensive analyses of small RNAs (sRNAs) from nine representative tomato accessions, we demonstrate that SVs substantially contribute to the dynamic expression of the three major classes of plant sRNAs: microRNAs (miRNAs), phased secondary short interfering RNAs (phasiRNAs), and 24-nt heterochromatic siRNAs (hc-siRNAs). Changes in the abundance of phasiRNAs and 24-nt hc-siRNAs likely contribute to the alteration of mRNA gene expression during tomatos recent evolution, particularly for genes associated with biotic and abiotic stress tolerance. We also observe that miRNA expression dynamics are associated with imprecise processing, alternative miRNA-miRNA* selections, and SVs. SVs mainly affect the expression of less-conserved miRNAs that do not have established regulatory functions or low abundant members in highly expressed miRNA families, highlighting different selection pressures on miRNAs compared to phasiRNAs and 24-nt hc-siRNAs. Our findings provide insights into plant sRNA evolution as well as SV-based gene regulation during crop domestication. Furthermore, our dataset provides a rich resource for mining the sRNA regulatory network in tomato.


title: Expansion microsopy reveals Plasmodium falciparum blood-stage parasites undergo anaphase with a chromatin bridge in the absence of mini-chromosome maintenance complex binding protein.

abstract: The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges, and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum.


title: The PhoPQ two-component system promotes carbapenem tolerance in Enterobacterales.

abstract:
Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of {beta}-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the {beta}-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulatory element mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance killing of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.

SignificanceTreating an infection with an antibiotic often fails, resulting in a tremendous public health burden. One understudied likely reason for treatment failure is the development of "antibiotic tolerance", the ability of bacteria to survive normally lethal exposure to an antibiotic. Here, we describe a molecular mechanism promoting tolerance. A bacterial stress sensor (PhoPQ) is activated in response to antibiotic (meropenem) treatment and consequently strengthens a bacterial protective "shell" to enhance survival. We also identify inhibitors of this mechanism, opening the door to developing compounds that help antibiotics work better against tolerant bacteria.


title: High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications.


title: Metabolite profiling reveals organ-specific flavone accumulation in Scutellaria and identifies a scutellarin isomer isoscutellarein 8-O-β-glucuronopyranoside

abstract: Scutellaria is a genus of plants containing multiple species with well-documented medicinal effects. S. baicalensis and S. barbata are among the best-studied Scutellaria species, and previous works have established flavones to be the primary source of their bioactivity. Recent genomic and biochemical studies with S. baicalensis and S. barbata have advanced our understanding of flavone biosynthesis in Scutellaria. However, as over several hundreds of Scutellaria species occur throughout the world, flavone biosynthesis in most species remains poorly understood. In this study, we analyzed organ-specific flavone profiles of seven Scutellaria species, including S. baicalensis, S. barbata and two species native to the Americas (S. wrightii to Texas and S. racemosa to Central and South America). We found that the roots of almost all these species produce only 4-deoxyflavones, while 4-hydroxyflavones are accumulated exclusively in their aerial parts. On the other hand, S. racemosa and S. wrightii also accumulated high levels of 4-deoxyflavones in their aerial parts, different with the flavone profiles of S. baicalensis and S. barbata. Furthermore, our metabolomics and NMR study identified the accumulation of isoscutellarein 8-O-{beta}-glucuronopyranoside, a rare 4-hydroxyflavone, in the stems and leaves of several Scutellaria species including S. baicalensis and S. barbata, but not in S. racemosa and S. wrightii. Distinctive organ-specific metabolite profiles among Scutellaria species indicate the selectivity and diverse physiological roles of flavones.


title: The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

abstract:
Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The  plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase.

One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.


title: T cells modulate the microglial response to brain ischemia

abstract:
Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. These findings substantiate the role of T cells in stroke with large impact on the cerebral inflammatory milieu by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.

SummaryThe crosstalk between brain infiltrating T cells and microglia in response to stroke remains elusive. Benakis et al. report that transcriptional signature of the stroke-associated microglia is reprogrammed by distinct T cell subpopulations. Engineered T cells overexpressing IL-10 administered four hours after stroke reinitiate microglial function inducing a pro-regenerative environment.


title: Variable freshwater influences on the abundance of Vibrio vulnificus in a tropical urban estuary

abstract:
To better understand the controls on the opportunistic human pathogen Vibrio vulnificus in warm tropical waters, we conducted a year-long investigation in the Ala Wai Canal, a channelized estuary in Honolulu, HI. The abundance of V. vulnificus as determined by qPCR of the hemolysin gene (vvhA), varied spatially and temporally nearly four orders of magnitude ([≤] 3 to 14,000 mL-1). Unlike in temperate and subtropical systems, temperatures were persistently warm (19-31{degrees}C) and explained little of the variability in V. vulnificus abundance. Salinity (1-36 ppt) had a significant, but non-linear, relationship with V. vulnificus abundance with highest vvhA concentrations (> 2,500 mL-1) observed only at salinities from 7 to 22 ppt. V. vulnificus abundances were lower on average in the summer dry season when waters were warmer but more saline. Highest canal-wide average abundances were observed during a time of modest rainfall when moderate salinities and elevated concentrations of reduced nitrogen species and silica suggested a groundwater influence. Distinguishing the abundances of two genotypes of V. vulnificus (C-type and E-type) suggest that C-type strains, which are responsible for most human infections, were usually less abundant (25% on average), but their relative contribution was greater at higher salinities, suggesting a broader salinity tolerance. Generalized regression models suggested up to 67% of sample-to-sample variation in log-transformed V. vulnificus abundance was explained (n = 202) using the measured environmental variables, and up to 97% of the monthly variation in canal-wide average concentrations (n = 13) was explained with the best subset of four variables.

IMPORTANCEOur data illustrate that, in the absence of strong seasonal variation in water temperature in the tropics, variation in salinity driven by rainfall becomes a primary controlling variable on V. vulnificus abundance. There is thus a tendency for a rainfall-driven seasonal cycle in V. vulnificus abundance that is inverted from the temperature-driven seasonal cycle at higher latitudes. However, stochasticity in rainfall and its non-linear, indirect effects on V. vulnificus concentration means that high abundances can occur at any location in the canal at any time of year, making it challenging to predict concentrations of this pathogen at high temporal or spatial resolution. Much of the variability in canal-wide average concentrations, on the other hand, was explained by a few variables that reflect the magnitude of freshwater input to the system, suggesting that relative risk of exposure to this pathogen could be predicted for the system as a whole.


title: Label-free Raman microspectroscopy for identifying virocells

abstract:
Raman microspectroscopy has been thoroughly used to assess growth dynamics and heterogeneity of prokaryotic cells. Yet, little is known about how the chemistry of individual cells changes during infection with lytic viruses, resulting in so-called virocells. Here, we investigate biochemical changes of bacterial and archaeal cells of three different species in laboratory cultures before and after addition of their respective viruses using single-cell Raman microspectroscopy. By applying multivariate statistics, we identified significant differences in the spectra of single cells and cells after addition of lytic phage (phi6) for Pseudomonas syringae. A general ratio of wavenumbers that contributed the greatest differences in the recorded spectra was defined as an indicator for virocells. Based on reference spectra, this difference is likely attributable to an increase in nucleic acid vs. protein ratio of virocells. This method proved also successful for identification of Bacillus subtilis cells infected with phi29 displaying a decrease in respective ratio but failed for archaeal virocells (Methanosarcina mazei with Methanosarcina Spherical Virus) due to autofluorescence. Multivariate and univariate analyses suggest that Raman spectral data of infected cells can also be used to explore the complex biology behind viral infections of bacteria. Using this method, we confirmed the previously described two-stage infection of P. syringaes phi6 and that infection of B. subtilis by phi29 results in a stress response within single cells. We conclude that Raman microspectroscopy is a promising tool for chemical identification of Gram-positive and Gram-negative virocells undergoing infection with lytic DNA or RNA viruses.

ImportanceViruses are highly diverse biological entities shaping many ecosystems across Earth. Yet, understanding the infection of individual microbial cells and the related biochemical changes remains limited. Using Raman microspectroscopy in conjunction with univariate and unsupervised machine learning approaches, we established a marker for identification of infected Gram-positive and Gram-negative bacteria. This non-destructive, label-free analytical method at single-cell resolution paves the way for future studies geared towards analyzing complex biological changes of virus-infected cells in pure culture and natural ecosystems.


title: Cryo-EM structure of a single-chain β1-adrenoceptor - AmpC β-lactamase fusion protein

abstract: The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors (GPCRs). Here, we explored the possibility of using a larger fusion protein, inserted into the third intracellular loop (ICL3) of {beta}1-adrenoceptor ({beta}1AR) via rigid chimeric helix fusions. The aim was to engineer a single-chain fusion protein that comprises sufficient mass and rigidity to allow single-particle cryo-EM data collection, without depending on binding proteins, such as G-proteins or nanobodies. Through parsing of the protein data bank (PDB), we identified the protein AmpC {beta}-lactamase as a suitable candidate. Both termini of this protein are -helical and the helices are antiparallel to each other. The distance between their centroids measures [~]11 [A]. Such a geometry is ideal to design extended chimeric helices with transmembrane (TM) helices 5 and 6 of {beta}1AR, and the insertion of the protein adds [~]39 kDa of mass to the receptor. We expressed the {beta}1AR - AmpC {beta}-lactamase fusion protein in mammalian cells. The binding of the antagonists propranolol and cyanopindolol to the purified fusion protein was confirmed by CPM-based thermofluor assays. The cryo-EM structure was solved to a nominal overall resolution of 3.6 [A] and the seven helix architecture and helix eight were clearly resolved. Superimposition of the structure with known X-ray crystal structures of {beta}1AR suggests that the protein is in its inactive conformation. The fusion protein described here provides a basis for high-throughput structure elucidation of class A GPCRs by cryo-EM for drug discovery research as well as for the elucidation of inactive state or wild-type GPCR structures. The fusion protein geometry theoretically fits a wide range of class A GPCRs and therefore can be applied to a multitude of receptors.

category: molecular biology


title: GLOBAL PATTERNS OF PLUMAGE COLOUR EVOLUTION IN ISLAND-LIVING PASSERIFORM BIRDS

abstract: Plumage coloration is an important trait involved communication and is shaped by a variety of ecological pressures. Island residency has the potential to change the evolutionary trajectory of plumage colour by differences in habitat and resources, or by altering predation pressure and social selection intensity. Latitude, island size, and isolation may further influence colour evolution by biasing colonization. Therefore, general patterns of plumage evolution are difficult to disentangle. We used phylogenetically controlled analyses to assess the influence of island residency on plumage colouration, by calculating chromaticity values from red, blue, green scores extracted from photos of Order Passeriformes birds. Importantly, we controlled for ecological factors hypothesized to influence colour evolution and assessed family-level effects. We found 1) colour varied between islands and mainlands in females, but not males, and both sexes were affected by several ecological factors; 2) patterns of colour evolution varied among families; 3) island size and distance to the mainland and other islands significantly influenced colour; and 4) interactions between ecological factors and latitude were consistently influenced colour, supporting a latitudinal gradient hypothesis. Our results indicate although island residency influences female colour evolution, a myriad of ecological factors drive plumage colour and the patterns vary among families.

category: ecology