title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

abstract: G protein-coupled receptors (GPCRs) regulate multiple processes ranging from cell growth and immune responses to neuronal signal transmission. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additional challenges exist to dissect cell type-specific responses when the same GPCR is expressed on different cells within the body. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that selectively bind their agonist clozapine-N-oxide (CNO) and mimic a GPCR-of-interest. We show that the chimeric DREADD-{beta}2-adrenergic receptor ({beta}2AR/ADRB2) triggers comparable responses to levalbuterol on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate {beta}2AR-mediated filopodia formation in microglia, a {beta}2AR-expressing immune cell that can drive inflammation in the nervous system. To further dissect microglial inflammation, we compared DREADD-{beta}2AR with two additionally designed DREADD-based chimeras mimicking GPR65 and GPR109A/HCAR2, both enriched in microglia. DREADD-{beta}2AR and DREADD-GPR65 modulate the inflammatory response with a similar profile as endogenously expressed {beta}2AR, while DREADD-GPR109A had no impact. Our DREADD-based approach allows investigation of cell type-dependent signaling pathways and function without known endogenous ligands.

category: synthetic biology


title: Targeting riboswitches with synthetic small RNAs for metabolic engineering

abstract:
Our growing knowledge of the diversity of non-coding RNAs in natural systems and our deepening knowledge of RNA folding and function have fomented the rational design of RNA regulators. Based on that knowledge, we designed and implemented a small RNA (sRNA) tool to target bacterial riboswitches and activate gene expression. The synthetic sRNA is suitable for regulation of gene expression both in cell-free and in cellular systems. It targets riboswitches to promote the antitermination folding regardless the cognate metabolite concentration. Therefore, it prevents transcription termination increasing gene expression up to 103-fold. We successfully used sRNA arrays for multiplex targeting of riboswitches. Finally, we used the synthetic sRNA to engineer an improved riboflavin producer strain. The easiness to design and construct, and the fact that the riboswitch-targeting sRNA works as a single genome copy, make it an attractive tool for engineering industrial metabolite-producing strains.

Graphical Abstract

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title: Immunological features that determine the strength of antibody responses to BNT162b2 mRNA vaccine against SARS-CoV-2

abstract: We analyzed peripheral blood mononuclear cells (PBMCs) of each 20 individuals with a high anti-SARS-CoV-2 antibody titer and a low antibody titer out of 1,774 healthcare workers who received BNT162b2 mRNA vaccine. A higher antibody titer was associated with the frequencies of naive and transitional B cells before vaccination. In addition, fold changes in the frequency of activated CD8+ T cells upon vaccination were correlated with the antibody titers.

category: immunology


title: The green tea catechin EGCG provides proof-of-concept for a pan-coronavirus entry inhibitor

abstract: The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized the serious threat to human health posed by emerging coronaviruses. Effective antiviral countermeasures are urgently needed as vaccine development and deployment against an emerging coronavirus takes time, even in the best-case scenario. The green tea catechin, epigallocatechin gallate (EGCG), has broad spectrum antiviral activity. We demonstrate here that EGCG prevents murine and human coronavirus infection and blocks the entry of lentiviral particles pseudotyped with spike proteins from highly pathogenic coronaviruses, as well as a bat coronavirus poised to emerge into humans. We show that EGCG treatment reduces coronavirus attachment to target cell surfaces. Our results demonstrate the potential for the development of pan-coronavirus attachment inhibitors to protect against current and future emerging coronaviruses.

category: microbiology


title: Antibiotics drive expansion of rare pathogens in a chronic infection microbiome model

abstract: Chronic (long-lasting) infections are globally a major and rising cause of morbidity and mortality. Unlike typical acute infections, chronic infections are ecologically diverse, characterized by the presence of a polymicrobial mix of opportunistic pathogens and human-associated commensals. To address the challenge of chronic infection microbiomes, we focus on a particularly well-characterized disease, cystic fibrosis (CF), where polymicrobial lung infections persist for decades despite frequent exposure to antibiotics. Epidemiological analyses point to conflicting results on the benefits of antibiotic treatment, and are confounded by the dependency of antibiotic exposures on prior pathogen presence, limiting their ability to draw causal inferences on the relationships between antibiotic exposure and pathogen dynamics. To address this limitation, we develop a synthetic infection microbiome model, and benchmark on clinical data. We show that, in the absence of antibiotics, the microbiome structure in a synthetic sputum medium is highly repeatable and dominated by oral commensals. In contrast, challenge with physiologically relevant antibiotic doses leads to substantial community perturbation characterized by multiple alternate pathogen-dominant states and enrichment of drug-resistant species. These results provide evidence that antibiotics can drive the expansion (via competitive release) of previously rare opportunistic pathogens and offer a path towards microbiome-informed conditional treatment strategies.


title: Allosteric activation of CwlD amidase activity by the GerS lipoprotein during Clostridioides difficile spore formation

abstract: Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. Spore germination depends on the degradation of the protective spore peptidoglycan layer known as the spore cortex. Cortex degradation is mediated by enzymes that recognize the spore-specific peptidoglycan modification, muramic-{partial}-lactam (MAL). In C. difficile, MAL synthesis depends on the activity of the CwlD amidase and the GerS lipoprotein, which directly binds CwlD. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind zinc stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to zinc, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of zinc co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.


title: Artemisinin-based hybrids produce intracellular proteasome inhibitors that overcome resistance in Plasmodium falciparum

abstract: Artemisinin resistant Plasmodium falciparum (Pf) is spreading despite combination chemotherapy (ACT). Here we report the design of artezomibs, single-molecule hybrids of an artemisinin and a Pf-selective proteasome inhibitor. Artezomibs exert a novel mode of action inside the malaria parasites. The artemisinin component covalently modifies parasite proteins, which become substrates of the Pf proteasome. The proteasomal degradation products that bear the proteasome inhibitor component of the hybrid then inhibit Pf proteasomes, including those with mutations that reduce binding affinity of the proteasome inhibitor component on its own. We demonstrated that artezomibs circumvent both artemisinin resistance conferred by Kelch13 polymorphism and resistance to the proteasome inhibitor associated with mutations in Pf proteasomes. This mode of action may enable the use of a single molecule with one pharmacokinetic profile to prevent the emergence of resistance.


title: L-form switching confers antibiotic, phage and stress tolerance in pathogenic Escherichia coli

abstract: The bacterial L-form is induced by exposure to cell wall targeting antibiotics or innate immune effectors such as lysozyme and is likely to be important in many human infections. Here, we demonstrate that the osmotically fragile L-form is a distinct physiological state in Escherichia coli that is highly tolerant of oxidative stress and resistant to powerful antibiotics and common therapeutic bacteriophages. L-forms quickly revert (<20h) to their cell-walled state after antibiotic withdrawal, with apparently normal physiology and with few or no changes in DNA sequence. T4-like phages that are obligately lytic in cell-walled E. coli preferentially pseudolysogenise their L-forms providing them with transient superinfection immunity. Our data indicate that L-form switching is a common response of pathogenic E. coli strains to cell wall-targeting antibiotics and that the most commonly used lytic bacteriophages are ineffective against them in this state.


title: CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

abstract: CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, including SPT5, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. Our phosphoproteomic analyses after CDK9 inhibition, using either DRB or an analog-sensitive CDK9 cell line, confirm the splicing factor SF3B1 as an additional key target of this kinase. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.

category: molecular biology


title: CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation.


title: Detecting Flavobacterial Fish Pathogens in the Environment Using High-Throughput Community Analysis

abstract:
Diseases caused by the fish pathogens Flavobacterium columnare and Flavobacterium psychrophilum are major contributors of preventable losses in the aquaculture industry. The persistent and difficult to control infections caused by these bacteria make timely intervention and prophylactic elimination of pathogen reservoirs important measures to combat these disease-causing agents. In the present study, we present two independent assays for detecting these pathogens in a range of environmental samples. Natural water samples were inoculated with F. columnare and F. psychrophilum cells, and pathogen levels were detected using Illumina MiSeq sequencing and droplet digital PCR. Both detection methods accurately identified pathogen-positive samples and showed good agreement in quantifying each pathogen. Additionally, the real-world application of these approaches was demonstrated using environmental samples collected at a rainbow trout aquaculture facility. These results show that both methods can serve as useful tools for surveillance efforts in aquaculture facilities, where the early detection of these flavobacterial pathogens may direct preventative measures to reduce disease occurrence.

ImportanceEarly detection of a deadly disease outbreak in a population can be the difference between mass fatality or mitigated effects. In the present study, we evaluated and compared two techniques for detecting economically impactful aquaculture pathogens. We demonstrate that one of these techniques, 16S rRNA gene sequencing using Illumina MiSeq technology, provides the ability to accurately detect two fish pathogens, F. columnare and F. psychrophilum, while simultaneously profiling the native microbial community. The second technique, droplet digital PCR, is commonly used for pathogen detection, and the results obtained using the assays we designed with this method served to validate those obtained using the MiSeq method. These two methods offer distinct advantages. The MiSeq method pairs pathogen detection and microbial community profiling to answer immediate and long-term fish health concerns, while droplet digital PCR method provides fast and highly sensitive detection that is useful for surveillance and rapid clinical responses.


title: Mapping the invisible chromatin transactions of prophase chromosome remodelling

abstract: We have used a combination of chemical genetics, chromatin proteomics and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 minutes following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.

category: cell biology


title: EXC-4/CLIC, Gα, and Rho/Rac signaling regulate tubulogenesis in C. elegans

abstract:
The Rho-family of small GTPases, which play crucial roles in development and disease, are regulated by many signal-transduction cascades, including G-protein-coupled receptor (GPCR)-heterotrimeric G-protein (G/{beta}/{gamma}) pathways. Using genetic approaches in C. elegans we identified a new role for G and Rho/Rac signaling in cell outgrowth during tubulogenesis and show that the Chloride Intracellular Channel (CLIC) protein EXC-4 is an evolutionarily-conserved player in this pathway. The gene exc-4 was identified by its role in tubulogenesis of the excretory canal (ExCa) cell--a unicellular tube required for osmoregulation and fluid clearance. We identified an exc-4 loss-of-function allele that affects an evolutionarily conserved residue in the C-terminus. Using this mutant we identified genetic interactions between exc-4, G, and Rho-family GTPases, defining novel roles for G-encoding genes (gpa-12/G12/13, gpa-7/Gi, egl-30/Gq, gsa-1/Gs) and the Rho-family members ced-10/Rac and mig-2/RhoG in ExCa outgrowth. EXC-4 and human CLICs have conserved functions in tubulogenesis, and CLICs and G-Rho/Rac signaling regulate tubulogenesis during blood vessel development. Therefore, our work defines a primordial role for EXC-4/CLICs in G-Rho/Rac-signaling during tubulogenesis.

One Sentence SummaryG and Rho/Rac signaling regulates EXC-4/CLIC-mediated cell outgrowth during tubulogenesis in C. elegans, linking elements of G-protein signaling to the enigmatic CLIC family of proteins.


title: A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium

abstract:
The most successful genetically encoded calcium indicators (GECIs) employ an intensity or intensiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a novel sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. A new engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. Additionally, the response of the calcium sensor is insensitive to pH between 6.2-9. As a result, the turquoise GECI enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentration with the turquoise GECI in single endothelial cells and human-derived organoids.

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title: Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

abstract: A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling pathway-regulated directed movement of cell clusters. OR is instructed by several signaling events, including the EGFR and Notch pathways, and planar cell polarity (PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification and differentiation. Here, we show that Abl kinase negatively regulates ommatidial rotation through its activity in the R3/R4 pair. Interestingly in wild-type, Abl is localized to apical junctional regions in R4 but not in R3 during OR, and this apical enrichment requires Notch signaling. We further demonstrate that Abl and Notch genetically interact during OR, and Abl co-immunoprecipitates in complexes with Notch in the developing eye disc. Perturbations of Abl interfere with adherens junction dynamics of the ommatidial preclusters, which are critical for the OR process. Taken together, our data suggest a model in which Abl kinase acts directly downstream of the Notch receptor in R4 to fine-tune OR via its input into adherens junction complexes.

category: developmental biology


title: Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum

abstract: For many important crops, including sorghum, the use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but more critically, by the ability to regenerate a fertile plant from the engineered cell through conventional tissue culture methods. Here, we report that Wuschel2 (Wus2)-enabled sorghum (Sorghum bicolor L.) transformation increases not only the efficiency of transgenic plant recovery, but also increases the observed efficiency of CRISPR/Cas-targeted genome editing within the population of T0 plants. Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased regeneration capacity, resulting in higher transformation efficiency across different sorghum genotypes. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrated up to an additional 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled, Agrobacterium-mediated transformation (compared to the control treatment with no Wus2), across various targeted loci in different sorghum genotypes. Wus2-enabled genome editing may be applicable to other crops and increasingly popular in planta transformation strategies.


title: One viral sequence for each host? - The neglected within-host diversity as the main stage of SARS-CoV-2 evolution

abstract: The standard practice of presenting one viral sequence for each infected individual implicitly assumes low within-host genetic diversity. It places the emphasis on the viral evolution between, rather than within, hosts. To determine this diversity, we collect SARS-CoV-2 samples from the same patient multiple times. Our own data in conjunction with previous reports show that two viral samples collected from the same individual are often very different due to the substantial within-host diversity. Each sample captures only a small part of the total diversity that is transiently and locally released from infected cells. Hence, the global SARS-CoV-2 population is a meta-population consisting of the viruses in all the infected hosts, each of which harboring a genetically diverse sub-population. Advantageous mutations must be present first as the within-host diversity before they are revealed as between-host polymorphism. The early detection of such diversity in multiple hosts could be an alarm for potentially dangerous mutations. In conclusion, the main forces of viral evolution, i.e., mutation, drift, recombination and selection, all operate within hosts and should be studied accordingly. Several significant implications are discussed.

category: evolutionary biology


title: N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease.

abstract:
BackgroundNormal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels.

MethodsTo enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread.

ResultsAnalysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission.

ConclusionsNeu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.

category: cancer biology


title: Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune pathways related to tissue injury

abstract:
Severe lung damage in COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways and genes present across the spectrum of histopathological damage in COVID-19 lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from areas of interest within well preserved post-mortem lung samples from three patients. Despite substantial inter-patient heterogeneity we discovered evidence for a common immune cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines including CXCL9, CXCL10 and CXCL11 which are known to promote the recruitment of CXCR3+ immune cells. The tumour necrosis factor (TNF) superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were found to be consistently upregulated in the areas with severe tissue damage. We used published spatial and single cell SARS-CoV-2 datasets to confirm our findings in the lung tissue from additional cohorts of COVID-19 patients. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.

One Sentence SummarySpatial analysis identifies IFN{gamma} response signatures as focal to severe alveolar damage in COVID-19 pneumonitis.


title: Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune circuits related to tissue injury


title: The molecular basis of extensively drug-resistant Salmonella Typhi isolates from pediatric septicemia patients

abstract: Sepsis is a syndromic response to infections and is becoming an emerging threat to the public health sector, particularly in developing countries. Salmonella Typhi (S. Typhi), the cause of typhoid fever, is one primary cause of pediatric sepsis in typhoid endemic areas. Extensively drug-resistant (XDR) S. Typhi is more common among pediatric patients, which is responsible for over 90% of the reported XDR typhoid cases, but the majority of antibiotic resistance studies available have been carried out using S. Typhi isolates from adult patients. Here, we characterized XDR S. Typhi isolates from a medium size cohort of pediatric typhoid patients to determine their antibiotic-resistance-related gene signatures associated with common treatment options to typhoid fever patients. This study informs the molecular basis of antibiotic-resistance among recent S. Typhi isolates from pediatric septicemia patients, therefore providing insights into the development of molecular detection methods and control strategies for XDR S. Typhi.


title: A ligated intestinal loop mouse model protocol to study the interactions of Clostridioides difficile spores with the intestinal mucosa during aging.

abstract: The interaction of the Clostridioides difficile spores with the intestinal mucosa contribute to the persistence and recurrence of the infection. Advanced age is one of the main risk factors to manifest C. difficile infection and recurrence. However, the interaction of C. difficile spores with the intestinal mucosa during aging has not been evaluated. In the present work, we provide a detailed protocol with all the critical information to perform an intestinal ligated loop. Using this technique in a mouse model, we evaluated C. difficile spore adherence and internalization to the ileum and colonic mucosa during aging. Consequently, our data suggest that spore internalization in the ileum and colonic mucosa is higher in elderly than in adults or young mice. Also, our data suggest that spore-adherence to the ileum and colonic mucosa decreases with aging.


title: Role of collagen-like protein BclA3 in the assembly of the exosporium layer of Clostridioides difficile spores.

abstract: Newly formed spores are essential for persistence of C. difficile in the host, transmission to a new susceptible host (Deakin et al., 2012b) and recurrence of CDI. BclA3 and BclA2 Spore surface proteins are expressed during sporulation under the control of mother-cell specific sigma factors of the RNA polymerase, SigE and SigK. Deletion of bclA3 leads to spores with an electron-dense exosporium layer that lacks bump-like structures in the electron-dense layer and hair-like projections, both structures typically found in the wild type spore. Therefore, in this work, we have addressed the role of the exosporium collagen-like BclA3 glycoprotein in the assembly of the exosporium layer. Immunogold labelling of BclA2CTD and BclA3CTD indicates that both proteins are located in the hairs, with BclA2 located outermost of BclA3. Absence of BclA3 leads to spores with no hair-like projections, and absence of bumps in thick exosporium spores, a phenotype also expressed in by the deletion of the collagen-like region of BclA3. Overall, these results provide insights into the role of BclA3 in the assembly of the exosporium layer of C. difficile spores.


title: Diversity and evolution of pigment types and the phycobilisome rod gene region of marine Synechococcus cyanobacteria

abstract: Synechococcus picocyanobacteria are ubiquitous and abundant photosynthetic organisms in the marine environment and contribute for an estimated 16% of the ocean net primary productivity. Their light-harvesting complexes, called phycobilisomes (PBS), are composed of a conserved allophycocyanin core from which radiates six to eight rods with variable phycobiliprotein and chromophore content. This variability allows Synechococcus to optimally exploit the wide variety of spectral niches existing in marine ecosystems. Seven distinct pigment types or subtypes have been identified so far in this taxon, based on the phycobiliprotein composition and/or the proportion of the different chromophores in PBS rods. Most genes involved in their biosynthesis and regulation are located in a dedicated genomic region called the PBS rod region. Here, we examined the variability of gene sequences and organization of this genomic region in a large set of sequenced isolates and natural populations of Synechococcus representative of all known pigment types. All regions start with a tRNA-PheGAA and some possess mobile elements including tyrosine recombinases, suggesting that their genomic plasticity relies on a tycheposon-like mechanism. Comparison of the phylogenies obtained for PBS and core genes revealed that the evolutionary history of PBS rod genes differs from the rest of the genome and is characterized by the co-existence of different alleles and frequent allelic exchange. We propose a scenario for the evolution of the different pigment types and highlight the importance of population-scale mechanisms in maintaining a wide diversity of pigment types in different Synechococcus lineages despite multiple speciation events.


title: Impacts of Mycoplasma agalactiae restriction-modification systems on pan-epigenome dynamics and genome plasticity

abstract: DNA methylation plays an important role in the biology of bacteria. Often associated with restriction modification (RM) systems, they also provide a defence against foreign DNA. Little is known regarding the methylome of the mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, single molecule real-time (SMRT) and bisulphite sequencing combined with whole-genome analysis identified 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems in Mycoplasma agalactiae, a ruminant pathogen and a model organism. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenome landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.


title: Gene editing in bacteria via SpCas9/sgRNA ribonucleoprotein complexes

abstract: Gene editing has been revolutionized by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas technology in a variety of organisms. In bacteria, however, CRISPR-Cas still holds many caveats such as high toxicity of Cas9 and off-target editing effects. In this work we develop a system for the incorporation of Cas9/single guide RNA ribonucleoprotein complexes in bacteria and their successful application for gene editing via homologous recombination. Targeting of a green fluorescent protein (GFP) reporter allows for easy verification of gene-edition via conversion to blue-fluorescent protein (BFP), mediated by the well characterized 196T > C (Tyr66His) mutation.


title: Multivalent interactions with RNA drive RNA binding protein recruitment and dynamics in biomolecular condensates in Xenopus oocytes

abstract: RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.


title: Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo

abstract: Stress granules are phase separated assemblies formed around mRNAs whose identities remain elusive. The techniques available to identify the RNA content of stress granules rely on their physical purification, and are therefore not suitable for single cells and tissues displaying cell heterogeneity. Here, we adapted TRIBE (Target of RNA-binding proteins Identified by Editing) to detect stress granule RNAs by fusing a stress granule RNA-binding protein (FMR1) to the catalytic domain of an RNA-editing enzyme (ADAR). RNAs colocalized with this fusion are edited, producing mutations that are detectable by sequencing. We first optimized the expression of this fusion protein so that RNA editing preferentially occurs in stress granules. We then show that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells, and in Drosophila tissues, such as 398 neuronal stress granule mRNAs encoding ATP binding, cell cycle and transcription factors. This new method opens the possibility to identify the RNA content of stress granules as well other RNA based assemblies in single cells derived from tissues.


title: Introduction of probiotic bacterial consortia promotes plant growth via impacts on the resident rhizosphere microbiome

abstract: Plant growth depends on a range of functions provided by their associated rhizosphere microbiome, including nutrient mineralization, hormone co-regulation and pathogen suppression. Improving the ability of plant associated microbiome to deliver these functions is thus important for developing robust and sustainable crop production. However, it is yet unclear how beneficial effects of probiotic microbial inoculants can be optimised and how they are mediated. Here, we sought to enhance the tomato plant growth by targeted introduction of probiotic bacterial consortia consisting of up to eight plant-associated Pseudomonas strains. We found that the effect of probiotic consortium inoculation was richness-dependent: consortia that contained more Pseudomonas strains, reached higher densities in the tomato rhizosphere and had clearer beneficial effects on multiple plant growth characteristics. Crucially, these effects were best explained by changes in the resident community diversity, composition and increase in the relative abundance of initially rare taxa, instead of introduction of plant-beneficial traits into the existing community along with probiotic consortia. Together, our results suggest that beneficial effects of microbial introductions can be driven indirectly through effects on the diversity and composition of resident plant rhizosphere microbiome.

category: ecology


title: The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor

abstract: Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community - the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of structural heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native mass spectrometry (MS) as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.

category: biochemistry


title: Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the Dos dormancy regulon

abstract:
Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KD = 5.64 {micro}M) but not the ferrous (Fe2+) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H2S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS-Fe3+. These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

Significance StatementHydrogen sulfide (H2S) is an important signaling molecule in eukaryotes and bacteria, and along with CO and NO, is an important part of host defense against Mycobacterium tuberculosis (Mtb). However, the mechanism(s) by which Mtb senses and responds to H2S is unknown. Here, we report that the Mtb heme sensor kinase DosS, a known sensor of CO and NO, is also a sensor of H2S. We found that H2S binds DosS in its ferric (Fe3+) state, which is considered as its inactive state, to induce the Dos dormancy regulon during infection. These data highlight the unusual capacity of Mtb to sense multiple gasotransmitters through a single sensing protein.


title: An artificial host system enables the obligate parasitic plant Cuscuta campestris to grow and complete its life cycle in vitro

abstract: Cuscuta campestris is an obligate parasitic plant that requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the uptake of water, nutrients and macromolecules. Research on Cuscuta is often complicated by host influences, but comparable systems for growing the parasite in the absence of a host do not exist. We developed an axenic method to grow C. campestris on an Artificial Host System (AHS). We evaluated the effects of nutrients and phytohormones on parasite haustoria development and growth. Haustorium morphology and gene expression were characterized. The AHS consists of an inert, fibrous stick that mimics a host stem, wicking water and nutrients to the parasite. It enables C. campestris to exhibit a parasitic habit and develop through all stages of its lifecycle, including production of new shoots and viable seeds. Phytohormones NAA and BA affect haustoria morphology, and increase parasite fresh weight and biomass. Gene expression in AHS haustoria reflect process similar to those in haustoria on actual host plants. The AHS is a methodological improvement for studying Cuscuta biology by avoiding specific host effects on parasite and giving researchers full control of the parasite environment.

category: plant biology


title: Clones and Clusters of Antimicrobial-Resistant Klebsiella from Southwestern Nigeria

abstract:
IntroductionKlebsiella pneumoniae is a World Health Organization high-priority antibiotic-resistant pathogen. However, little is known about the population structure and evolution of Klebsiella circulating in Nigeria.

MethodsWe performed whole genome sequencing (WGS) of 141 Klebsiella isolated between 2016 and 2018 from clinical specimens at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. We conducted in silico multilocus sequence typing, AMR gene, virulence gene, plasmid, and K and O loci profiling, as well as phylogenetic analyses, using publicly available tools and Nextflow pipelines.

ResultsPhylogenetic analysis revealed that the majority of the 134 K. pneumoniae and 5 K. quasipneumoniae isolates from Nigeria characterized are closely related to globally disseminated multidrug-resistant clones. Of the 39 K. pneumoniae sequence types (STs) identified, the most common were ST307 (15%), ST5241 (12%), ST15 (~9%), and ST25 (~6%). ST5241, one of 10 novel STs detected, is a single locus variant of ST636 carrying dfrA14, tetD, qnrS, and oqxAB resistance genes. The extended-spectrum {beta} lactamase (ESBL) gene blaCTX_M-15 was seen in 72 % of K. pneumoniae genomes, while 8% encoded a carbapenemase. Four likely outbreak clusters from one facility, within STs 17, 25, 307, and 5241, were ESBL but not carbapenemase-bearing clones.

ConclusionThis study uncovered known and novel K. pneumoniae lineages circulating in Nigeria that include multidrug-resistant ESBL producers. Carbapenemase-producing isolates remain uncommon. WGS retrospectively identified outbreak clusters, pointing to the value of genomic approaches in AMR surveillance for improving infection prevention and control in Nigerian hospitals.

summaryWe performed whole genome sequencing (WGS) of 141 Klebsiella isolated in 2016-2018 at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. This study uncovered known and novel K. pneumoniae lineages circulating in Nigeria that include multidrug-resistant ESBL producers.

FUNDINGThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [16/136/111: NIHR Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance].

This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. INO is an African Research Leader supported by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement that is also part of the EDCTP2 program supported by the European Union. The funders had no role in the content, crafting or submission of this paper. The views expressed in this publication are those of the authors and not necessarily those of the funders or their affiliates.

CONFLICT OF INTERESTThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

category: genomics


title: The AMIGO1 adhesion protein modulates movement of Kv2.1 voltage sensors

abstract:
Kv2 voltage-gated potassium channels are modulated by AMIGO neuronal adhesion proteins. Here, we identify steps in the conductance activation pathway of Kv2.1 channels that are modulated by AMIGO1 using voltage clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines. AMIGO1 speeds early voltage sensor movements and shifts the gating charge-voltage relationship to more negative voltages. The gating charge-voltage relationship indicates that AMIGO1 exerts a larger energetic effect on voltage sensor movement than apparent from the midpoint of the conductance-voltage relationship. When voltage sensors are detained at rest by voltage sensor toxins, AMIGO1 has a greater impact on the conductance-voltage relationship. Fluorescence measurements from voltage sensor toxins bound to Kv2.1 indicate that with AMIGO1, the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. We conclude that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors.

Statement of SignificanceKv2 potassium channels activate a potassium conductance that shapes neuronal action potentials. The AMIGO family of adhesion proteins modulate activation of Kv2 conductances, yet, which activation steps are modified is unknown. This study finds that AMIGO1 destabilizes the earliest resting conformation of the Kv2.1 voltage sensors to promote activation of channel conductance.

category: biophysics


title: The AMIGO1 adhesion protein activates Kv2.1 voltage sensors


title: Phytochrome gene expression studies: PHYE is required for FR-induced expression of PHYA and PHYD suppresses expression of PHYA

abstract: Arabidopsis has five phytochrome (PHY) genes for sensing the Red:Far Red (R:FR) ratio in ambient light, of which PHYA has an established role in responses to FR. To study whether and how PHYs may influence each others transcription, PHY-Luciferase reporter plants (pPHYA:LUC, pPHYB:LUC, pPHYC:LUC, pPHYD:LUC and pPHYE:LUC) were constructed. Subsequently, reporter lines representative for each PHY were crossed into each of the five single phy-mutant backgrounds. Reporter activities in WT and phy mutant was studied under diurnal mixed (R, B, FR), R, FR or B LED light in seedling or rosette plants. Both pPHYA:LUC and pPHYB:LUC show strong induction under FR. Full FR upregulation of both PHYA and PHYB is dependent on PHYE, identifying PHYE as a novel sensor for FR light responses. Results also show that PHYA expression is strongly suppressed by PHYD. Results were confirmed for expression of endogenous PHYA and PHYB, albeit with different dynamics compared to the LUC reporters. Profiling of pPHYA:LUC and pPHYB:LUC reporters suggest gating of FR responses. Manipulation of PHY expression levels by FR may provide a novel basis for manipulating plant growth in controlled environments.


title: Analysis of scientific journalism in Nature reveals gender and regional disparities in coverage

abstract:
Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations.

category: scientific communication and education


title: Analysis of science journalism reveals gender and regional disparities in coverage


title: Integrative analysis of multi-omics reveals gene regulatory networks across brain regions from risk variants to phenotypes of Alzheimer's disease and Covid-19

abstract:
BackgroundGenome-wide association studies have found many genetic risk variants associated with Alzheimers disease (AD). However, how these risk variants affect deeper phenotypes such as disease progression and immune response remains elusive. Also, our understanding of cellular and molecular mechanisms from disease risk variants to various phenotypes is still limited. To address these problems, we performed an integrative multi-omics analysis of genotype, transcriptomics, and epigenomics for revealing gene regulatory mechanisms from disease variants to AD phenotypes.

MethodFirst, given the population gene expression data of a cohort, we construct and cluster its gene co-expression network to identify gene co-expression modules for various AD phenotypes. Next, we predict transcription factors (TFs) regulating co-expressed genes and AD risk SNPs that interrupt TF binding sites on regulatory elements. Finally, we construct a gene regulatory network (GRN) linking SNPs, interrupted TFs, and regulatory elements to target genes and gene modules for each phenotype in the cohort. This network thus provides systematic insights into gene regulatory mechanisms from risk variants to AD phenotypes.

ResultsOur analysis predicted GRNs in three major AD-relevant regions: Hippocampus, Dorsolateral Prefrontal Cortex (DLPFC), Lateral Temporal Lobe (LTL). Comparative analyses revealed cross-region-conserved and region-specific GRNs, in which many immunological genes are present. For instance, SNPs rs13404184 and rs61068452 disrupt SPI1 binding and regulation of AD gene INPP5D in the Hippocampus and LTL. However, SNP rs117863556 interrupts bindings of REST to regulate GAB2 in DLPFC only. Driven by emerging neuroinflammation in AD, we used Covid-19 as a proxy to identify possible regulatory mechanisms for neuroimmunology in AD. To this end, we looked at the GRN subnetworks relating to genes from shared AD-Covid pathways. From those subnetworks, our machine learning analysis prioritized the AD-Covid genes for predicting Covid-19 severity. Decision Curve Analysis also validated our AD-Covid genes outperform known Covid-19 genes for classifying severe Covid-19 patients. This suggests AD-Covid genes along with linked SNPs can be potential novel biomarkers for neuroimmunology in AD. Finally, our results are open-source available as a comprehensive functional genomic map for AD, providing a deeper mechanistic understanding of the interplay among multi-omics, brain regions, gene functions like neuroimmunology, and phenotypes.


title: Predicting gene regulatory networks from multi-omics to link genetic risk variants and neuroimmunology to Alzheimers disease phenotypes


title: Omics-based label-free metabolic flux inference reveals dysregulation of glucose metabolism in liver associated with obesity

abstract:
Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify changes in metabolic flux, and to quantify contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in liver.

HighlightsO_LIWe created OMELET to infer metabolic flux and its regulation from multi-omic data.
C_LIO_LIGluconeogenic and pyruvate cycle fluxes increased in fasting ob/ob mice.
C_LIO_LITranscripts increases mediated the increase in gluconeogenic fluxes in ob/ob mice.
C_LIO_LIIncreases in transcripts and substrates enhanced pyruvate cycle flux in ob/ob mice.
C_LI

category: systems biology


title: Multi-omics-based label-free metabolic flux inference reveals obesity-associated dysregulatory mechanisms in liver glucose metabolism


title: A streamlined CRISPR workflow to introduce mutations and generate isogenic iPSCs for modeling amyotrophic lateral sclerosis

abstract: Amyotrophic lateral sclerosis (ALS) represents a complex neurodegenerative disorder with significant genetic heterogeneity. To date, both the genetic etiology and the underlying molecular mechanisms driving this disease remain poorly understood, although in recent years a number of studies have highlighted a number of genetic mutations causative for ALS. With these mutations pointing to potential pathways that may be affected within individuals with ALS, having the ability to generate human neurons and other disease relevant cells containing these mutations becomes even more critical if new therapies are to emerge. Recent developments with the advent of induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing fields gave us the tools to introduce or correct a specific mutation at any site within the genome of an iPSC, and thus model the specific contribution of risk mutations. In this study we describe a rapid and efficient way to either introduce a mutation into a control line, or to correct a mutation, generating an isogenic control line from patient-derived iPSCs with a given mutation. The mutations introduced were the G93A mutation into SOD1 or H517Q into FUS, and the mutation corrected was a patient iPSC line with I114T in SOD1. A combination of small molecules and growth factors were used to guide a stepwise differentiation of the edited cells into motor neurons in order to demonstrate that disease-relevant cells could be generated for downstream applications. Through a combination of iPSCs and CRISPR editing, the cells generated here will provide fundamental insights into the molecular mechanisms underlying neuron degeneration in ALS.

category: neuroscience


title: Structural insights into ligand recognition and activation of the melanocortin-4 receptor

abstract: Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning receptor activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric Gs protein stimulated by the endogenous peptide -MSH, FDA-approved drugs afamelanotide (Scenesse) and bremelanotide (Vyleesi), and selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.


title: Deconvolution of the epigenetic age discloses distinct inter-personal variability in epigenetic aging patterns

abstract:
BackgroundThe epigenetic age can now be extrapolated from one of several epigenetic clocks, which are based on age-related changes in DNA methylation levels at specific multiple CpG sites. Accelerated aging, calculated from the discrepancy between the chronological age and the epigenetic age, has shown to predict morbidity and mortality rate. We assumed that deconvolution of epigenetic age to its components could shed light on the diversity of epigenetic, and by inference, on inter-individual variability in the causes of biological aging.

ResultsUsing the Horvath original epigenetic clock, we identified several CpG sites linked to distinct genes that quantitatively explain much of the interpersonal variability in epigenetic aging, with CpG sites related to secretagogin and malin being the most variable. We show that equal epigenetic age in different subjects can result from variable contribution size of the same CpG sites to the total epigenetic age. In a healthy cohort, the most variable CpG sites are responsible for accelerated and decelerated epigenetic aging, relative to chronological age.

ConclusionsOf the 353 CpG sites that form the basis for the Horvath epigenetic age, we have found the CpG sites that are responsible for accelerated and decelerated epigenetic aging in healthy subjects. However, the relative contribution of each site to aging varies between individuals, leading to variable personal aging patterns. Our findings pave the way to form personalized aging cards allowing the identification of specific genes related to CpG sites, as aging markers, and perhaps treatment of these targets in order to hinder undesirable age drifting.


title: Ecological memory of prior nutrient exposure in the human gut microbiome

abstract:
Many ecosystems retain an ecological memory of past conditions that affects responses to future stimuli. However, it remains unknown what mechanisms and dynamics may govern such a memory in microbial communities. Here, in both a human dietary intervention cohort and an artificial gut, we show that the human gut microbiome encodes a memory of past carbohydrate exposures. Changes in the relative abundance of primary degraders were sufficient to enhance metabolism, and these changes were mediated by transcriptional changes within hours of initial exposure. We further found that ecological memory of one carbohydrate species impacted metabolism of others. These findings demonstrate that the human gut microbiomes metabolic potential reflects dietary exposures over preceding days and changes within hours of exposure to a novel nutrient.

One Sentence SummaryRecent nutrient exposures are encoded into the structure and activity of human gut microbial communities, which enables more efficient future metabolic responses.


title: New insights from the combined discrimination of modern/ancient genome-wide shared alleles and haplotypes: Differentiated demographic history reconstruction of Tai-Kadai and Sinitic people in South China

abstract: Southern China was a region with mixed rice-millet farming during the Middle Neolithic period and also suggested to be the homeland of Tai-Kadai-speaking (TK) people. The archaeological evidence of animal and plant domestication has demonstrated that southern Chinese rice agriculturalists dispersed from the Yangtze River basin with the dissemination of TK, Austroasiatic (AA), Austronesian (AN) and Hmong-Mien (HM) languages. However, the formations of the inland TK-speaking people, central/southern Han Chinese and their relationships with Neolithic farmers from the Yangtze and Yellow Rivers (YR) basins are far from clear due to the limited sampling of South China. Here, we revealed the spatiotemporal demographic history of southern China by analyzing newly generated genome-wide data of 70 southeastern mainland TK speakers including Dong, Gelao and Bouyei and 45 southwestern Han Chinese together with comprehensive modern/ancient reference datasets. Southwest Han Chinese and Gelao demonstrated a closer genomic affinity to Neolithic YR farmers, while inland TKs (Dong and Bouyei) demonstrated a closer genomic affinity to coastal TK/AN-speaking islanders and Neolithic Yangtze farmers and their descendants. The shared genetic drift between inland TK/AN speaker highlighted a common origin of AN/TK groups, which may be descended from Tanshishan people or their predecessors (Xitoucun). Additionally, we found that inland TK/Sinitic could be modelled as an admixture of ancestral northern East Asian (ANEA) and ancestral southern East Asian (ASEA) sources with different proportions, in which the ANEA was phylogenetically closer to Neolithic millet farmers deriving from the YR Basin and the ASEA was phylogenetically closer to Coastal Neolithic-to-modern southern East Asians. Finally, we discovered genetic differentiation among TK people from southern China and Southeast Asia and obvious substructures between northern and southern inland Chinese TK people. The observed patterns of the spatiotemporal distribution of the northern and southern East Asian lineages in Central/southern China were also compatible with the scenario of bi-directional gene flow events from ANEA and ASEA. Conclusively, multiple lines of genomic evidence indicated millet farmers deriving from the YR basin and rice farmers deriving from the Yangtze River basin substantially contributed to the present-day mainland TK speakers and Central/southern Han Chinese, and formed the modern dual genetic admixture profile.

category: genetics


title: VarGenius-HZD allows accurate detection of rare homozygous or hemizygous deletions in targeted sequencing leveraging breadth of coverage

abstract:
MotivationHomozygous deletions (HDs) may be the cause of rare diseases and cancer and their discovery in targeted sequencing is a challenging task. Different tools have been developed to disentangle HD discovery but a sensitive caller is still lacking.

ResultsWe present VarGenius-HZD, a sensitive and scalable algorithm that leverages breadth-of-coverage for the detection of rare homozygous and hemizygous single-exon deletions (HDs). To assess its effectiveness we detected both real and synthetic rare HDs in fifty exomes from the 1000 Genomes Project obtaining higher sensitivity in comparison with state-of-the-art algorithms which missed at least one event each. We then applied our tool on targeted sequencing data from patients with Inherited Retinal Dystrophies and solved five cases that still lacked a genetic diagnosis.

Availability and implementationWe provide VarGenius-HZD either stand-alone or integrated within our recently developed software enabling the automated selection of samples using the internal database. Hence, it could be extremely useful for both diagnostic and research purposes. Our tool is available under GNU General Public License, version 3 at: https://github.com/frankMusacchia/VarGenius-HZD

Contact: francesco.musacchia@iit.it

Supplementary information is available online.


title: MungeSumstats: A Bioconductor package for the standardisation and quality control of many GWAS summary statistics

abstract:
SummaryGenome-wide association studies (GWAS) summary statistics have popularised and accelerated genetic research. However, a lack of standardisation of the file formats used has proven problematic when running secondary analysis tools or performing meta-analysis studies. To address this issue, we have developed MungeSumstats, a Bioconductor R package for the standardisation and quality control of GWAS summary statistics. MungeSumstats can handle the most common summary statistic formats, including variant call format (VCF) producing a reformatted, standardised, tabular summary statistic file, VCF or R native data object.

ContactAlan Murphy: a.murphy@imperial.ac.uk, Nathan Skene: n.skene@imperial.ac.uk

Availability and implementationMungeSumstats is available on Bioconductor (v 3.13) and can also be found on Github at: https://neurogenomics.github.io/MungeSumstats

Supplementary informationThe analysis deriving the most common summary statistic formats is available at: https://al-murphy.github.io/SumstatFormats


title: Analysis of the H-Ras mobility pattern in vivo shows cellular heterogeneity inside epidermal tissue

abstract:
Over the last two decades, developments in single-molecule microscopy (SMM) have enabled imaging and tracking of individual, fluorescently labelled proteins in biological systems, and most of these studies have focused on the analysis of protein mobility patterns inside cultured cells. In the present study, SMM was applied in vivo, using the zebrafish embryo model. We studied the protein dynamics of the membrane protein H-Ras, mutants of this protein, and its membrane-anchoring domain, C10H-Ras, in epidermal cells of living two-day-old embryos, using a total internal reflection fluorescence microscopy (TIRFM) setup. For all proteins studied, our results consistently confirm the presence of a fast- and a slow-diffusing subpopulations of molecules, which both confine to microdomains within the plasma membrane. Although the mobility patterns of H-Ras, mutants of this proteins and its membrane-anchoring domain were remarkably similar, the constitutively active H-Ras mutant, H-RasV12, exhibited significantly higher diffusion rates than the wild-type H-Ras and its inactive mutant, H-RasN17. Ultimately, we characterized variability in our data obtained using the zebrafish embryo model and demonstrated that differences between cells within the same embryo are the largest source of variation in our data. Our findings are in line with a model in which the cellular architecture of individual cells within a tissue determine the mobility of H-Ras proteins anchored in the plasma membrane cytoplasmic leaflet. Thereby, our results underline the growing importance of performing SMM imaging in vivo in order to better understand factors influencing the protein dynamics in an intact living organism.

SUMMARY STATEMENTBy application of single-molecule microscopy to living zebrafish embryos, factors altering the in vivo dynamics of H-Ras proteins in epidermal cells were analyzed.


title: Train-the-Trainer as an Effective Approach to Building Global Networks of Experts in Genomic Surveillance of AMR

abstract:
Advanced genomics and sequencing technologies are increasingly becoming critical for global health applications such as pathogen and antimicrobial resistance (AMR) surveillance. Limited resources challenge capacity development in low- and middle-income countries (LMICs), with few countries having genomics facilities and adequately trained staff. Training research and public health experts who are directly involved in the establishment of such facilities offers an effective, but limited, solution to a growing need. Instead, training them to impart their knowledge and skills to others provides a sustainable model for scaling up the much needed capacity and capability for genomic sequencing and analysis locally with global impact. We designed and developed a Train-the-Trainer course integrating pedagogical aspects with genomic and bioinformatics activities. The course was delivered to 18 participants from 12 countries in Africa, Asia, and Latin America. A combination of teaching strategies culminating in a group project created a foundation for continued development at home institutions. Upon follow-up after 6 months, at least 40% of trainees had initiated training programs and collaborations to build capacity at local, national, and regional level. This work provides a framework for implementing a training and capacity building program for the application of genomics tools and resources in AMR surveillance.

40-word summaryThis work provides a framework for implementing a training and capacity building program for the application of genomics tools and resources in AMR surveillance. We outline a Train-the-Trainer course integrating pedagogical aspects with genomic and bioinformatics activities.


title: Constitutive IFNα protein production in bats

abstract: Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFN protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but was not associated with age, sex, or health status suggesting constitutive IFN protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.

category: zoology


title: Identification of the key role of white matter alteration in the pathogenesis of Huntington's Disease

abstract:
Pathogenesis of the inherited neurodegenerative disorder Huntingtons Disease (HD) is complex and progressive, with a long presymptomatic phase in which subtle changes occur in the brain of gene carriers up to 15 years before the onset of symptoms. Thus, there is a need of early, functional biomarker to better understand disease progression and to evaluate treatment efficacy far from onset. In particular, recent studies have shown that white matter may be affected early in HD. In this study, we scanned longitudinally Ki140CAG mice with structural MRI, Diffusion Tensor Imaging (DTI), Chemical Exchange Saturation Transfer of glutamate (gluCEST) and Magnetization Transfer (MT) imaging, in order to assess white matter integrity over the life of this very progressive mouse model. Our results show early defects of diffusion properties in the anterior part of the corpus callosum, preceding gluCEST defects in the same region (-10.8% at 8 months, -19% at 12 months) that spread to adjacent regions. At 12 months, frontal (-7.3%) and piriform (-16.7%) cortices showed reduced gluCEST, as well as the pallidum (-21.0%). MT imaging showed reduced signal in the septum (-21.7%) at 12 months. Cortical and striatal atrophy then appear at 18 months. Vulnerability of the striatum and motor cortex, combined with alterations of anterior corpus callosum, seems to point out the pivotal role of white matter, in the pathogenesis of HD and the pertinence of gluCEST and DTI as biomarkers in HD.

Highlights- A knock-in mouse model of Huntingtons disease is longitudinally characterized
- A multimodal MRI protocol is performed to identify biomarkers of the disease
- The white matter plays a pivotal role in the pathogenesis of the disease
- The cortico-striatal pathway seems particularly vulnerable in Huntingtons disease


title: The functional properties of synapses made by regenerated axons across spinal cord lesion sites

abstract:
While the anatomical properties of regenerated axons across spinal cord lesion sites have been studied extensively, little is known of how the functional properties of regenerated synapses compare to those in unlesioned animals. This comparison has been performed here in the lamprey, a model system for spinal injury research, in which functional locomotor recovery after spinal cord lesions is associated with axonal regeneration across the lesion site.

Regenerated synapses below the lesion site did not differ to synapses from unlesioned axons with respect to the amplitude and duration of single excitatory postsynaptic potentials (EPSPs). They also showed the same activity-dependent depression over spike trains. However, regenerated synapses did differ to unlesioned synapses as the estimated number of synaptic vesicles was greater and there was evidence for an increased postsynaptic quantal amplitude. For axons above the lesion site, the amplitude and duration of single synaptic inputs also did not differ significantly to unlesioned animals. However, in this case there was evidence of a reduction in release probability and inputs facilitated rather that depressed over spike trains.

Synaptic inputs from single regenerated axons below the lesion site thus do not increase in amplitude to compensate for the reduced number of descending axons after functional recovery. However, the postsynaptic input is maintained at the unlesioned level using different synaptic properties. Conversely, the facilitation from the same initial amplitude above the lesion site will make the synaptic input over spike trains functionally stronger. This may help to increase propriospinal activity across the lesion site to compensate for the lesion-induced reduction in supraspinal inputs.


title: Genetics of tibia bone properties of crossbred commercial laying hens in different housing systems

abstract:
Osteoporosis and bone fractures are a severe problem for the welfare of laying hens, with genetics and environment, such as housing system, each making substantial contributions to bone strength. In this work, we performed genetic analyses of bone strength, bone mineral density and bone composition, as well as body weight, in 860 commercial crossbred laying hens from two different companies, kept in either furnished cages or floor pens. We compared bone traits between housing systems and crossbreds, and performed a genome-wide association study of bone properties and body weight.

As expected, the two housing systems produced a large difference in bone strength, with layers housed in floor pens having stronger bones. These differences were accompanied by differences in bone geometry, mineralisation and chemical composition. Genome-scans either combining or independently analysing the two housing systems revealed no genome-wide significant loci for bone breaking strength. We detected three loci for body weight that were shared between the housing systems on chromosomes 4, 6 and 27 (either genome-wide significant or suggestive when the housing systems were analysed individually) and these coincide with associations for bone length.

In summary, we found substantial differences in bone strength, content and composition between hens kept in floor pens and furnished cages that could be attributed to greater physical activity in pen housing. We found little evidence for large-effect loci for bone strength in commercial crossbred hens, consistent with a highly polygenic architecture for bone strength in the production environment. The lack of consistent genetic associations between housing systems in combination with the differences in bone phenotypes support gene-by-environment interactions with housing system.


title: A novel binary k-mer approach for classification of coding and non-coding RNAs across diverse species

abstract: Classification among coding (CDS) and non-coding RNA (ncRNA) sequences is a challenge and several machine learning models have been developed for the same. Since the frequency of curated coding sequences is many-folds as compared to that of the ncRNAs, we devised a novel approach to work with the complete datasets from fifteen diverse species. In our proposed novel binary approach, we replaced all the  As and  Ts with  0s and  Gs and  Cs with  1s to obtain a binary form of coding and ncRNAs. The k-mer analysis of these binary sequences revealed that the frequency of binary patterns among the coding and ncRNAs can be used as features to distinguish among them. Using insights from these distinguishing frequencies, we used k-nearest neighbour classifier to classify among them. Our strategy is not only time-efficient but leads to significantly increased performance metrics including Matthews correlation coefficient (MCC) for some species like P. paniscus, M. mulatta, M. lucifugus, G. gallus, C. japonica, C. abingdonii, A. carolinensis, D. melanogaster and C. elegans when compared with the conventional ATGC approach. Additionally, we also show that the values of MCC obtained for diverse species tested on the model based on H. sapiens correlated with the geological evolutionary timeline thereby further strengthening our approach. Therefore, we propose that CDS and ncRNAs can be efficiently classified using "2-character" frequency as compared to "4-character" frequency of ATGC approach. Thus, our highly efficient binary approach can replace the more complex ATGC approach successfully.


title: Molecular Determinants of Ligand Residence in Galectin

abstract:
The recognition of carbohydrates by lectins play key roles in diverse cellular processes such as cellular adhesion, proliferation and apoptosis which makes it a promising therapeutic target against cancers. One of the most functionally active lectins, galectin-3 is distinctively known for its specific binding affinity towards {beta}-galactoside. Despite the prevalence of high-resolution crystallographic structures, the mechanistic basis and the molecular determinants of the sugar recognition process by galectin-3 are currently elusive. Here we address this question by capturing the complete dynamical binding process of human galectin-3 with its native ligand N-acetyllactosamine (LacNAc) and one of its synthetic derivatives by unbiased Molecular Dynamics simulation. In our simulations, both the natural ligand LacNAc and its synthetic derivative, initially solvated in water, diffuse around the protein and eventually recognise the designated binding site at the S-side of galectin-3, in crystallographic precision and identifies key metastable intermediate ligand-states around the galectin on their course to eventual binding. The simulations highlight that the origin of the experimentally observed multi-fold efficacy of synthetically designed ligand-derivative over its native natural ligand LacNAc lies in the derivatives relatively longer residence time in the bound pocket. A kinetic analysis demonstrates that the LacNAc-derivative would be more resilient compared to the parent ligand against unbinding from the protein binding site. In particular, the analysis identifies that interactions of the binding pocket residues Trp181, Arg144 and Arg162 with the tetrafuorophenyl ring of the derivative as the key determinant for the synthetic ligand to latch into the pocket.

Graphical Abstract

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title: An Empirical Energy Landscape Reveals Mechanism of Proteasome in Polypeptide Translocation

abstract: The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits1,2. How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is not known. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasomes heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions were widely consistent with experimental observations in this and previous studies and suggested novel features of the mechanism of proteasomal ATPase. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding state. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.


title: Divergent functional and conformational outcomes in an ion channel-peptide interaction

abstract: Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.


title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition


title: I will teach you here or there, I will try to teach you anywhere: perceived supports and barriers for emergency remote teaching during COVID-19 pandemic

abstract:
BackgroundDue to the COVID-19 pandemic, many universities moved to emergency remote teaching (ERT). This allowed institutions to continue their instruction despite not being in person. However, ERT is not without consequences. For example, students may have inadequate technological supports, such as reliable internet and computers. Students may also have poor learning environments at home and may need to find added employment to support their families. Additionally, there were consequences to faculty. It has been shown that female instructors are more disproportionately impacted in terms of mental health issues and increased domestic labor.

This research aims to investigate instructors and students perceptions of their transition to ERT. Specifically, we wanted to: O_LIIdentify supports and barriers during the transition to ERT
C_LIO_LICompare instructors experiences with the student experiences
C_LIO_LIExplore these supports and barriers within the context of social presence, teaching presence, and/or cognitive presence during ERT as well as how these supports and barriers relate to scaffolding in emergency remote courses
C_LI

DesignUsing grounded theory techniques, we applied two-cycle, qualitative analyses to assess the instructor transcripts. In first-cycle analysis, we used open coding to develop initial ideas from the data. We then used second cycle coding to generate categories with definitions and criteria agreed upon during discussion-based consensus building. Finally, these categories and descriptions were used to code student survey data.

Analyses/InterpretationsInstructors identified twice as many barriers as supports in their teaching during the transition to ERT and identified casual and formal conversations with colleagues as valuable supports. Emerging categories for barriers consisted of academic integrity concerns as well as technological difficulties. Similarly, students identified more barriers than supports in their learning during the transition to ERT. More specifically, students described pre-existing course structure, classroom technology, and community as best supporting their learning. Barriers that challenged student learning included classroom environment, student availability, and student emotion and comfort.

ContributionTogether, this research will help us understand supports and barriers to teaching and learning during the transition to ERT. This understanding can help us better plan and prepare for future emergencies, particularly at MSIs, where improved communication and increased access to resources for both students and instructors are key.


title: Emotion increases the similarity between neural representations of pictures, but not their perceived similarity.

abstract: Stimuli that evoke the same feelings can nevertheless look different and have different semantic meanings. Although we know much about the neural representation of emotion, the neural underpinnings that govern judgements of emotional similarity are unknown. One possibility is that the same brain regions will represent similarity between emotional and neutral stimuli, perhaps with different strengths. Alternatively, emotional similarity could be coded in separate regions, possibly those known to express emotional valence and arousal preferentially. In behaviour, the extent to which people consider similarity along the emotional dimension when they evaluate the overall similarity between stimuli has never been investigated. While the emotional features of stimuli may dominate explicit ratings of similarity, it is also possible that people neglect the emotional dimension as irrelevant. We contrasted these hypotheses with two measures of similarity and two different databases of complex negative and neutral pictures, the second of which afforded exquisite control over semantic and visual attributes. Emotion increased neural similarity in a set of regions that represented both emotional and neutral stimuli, including the inferior temporal cortex, the fusiform face area, and the precuneus. Emotion also increased neural similarity in early visual cortex, anterior insula and dorsal anterior cingulate cortex, despite no increase in BOLD-signal amplitudes in these regions. Despite the stronger neural similarity between emotional stimuli, participants rated pictures taken from two distinct emotional categories as equally similar. These results contribute to our understanding of how emotion is represented within a general conceptual workspace.


title: The Physiometrics of Inflammatory Proteins and Implications for Medical and Psychiatric Research: Investigation of Empirically-informed Inflammatory Composites

abstract: Most research testing the association between inflammation and health outcomes (e.g., heart disease, diabetes, depression) has focused on individual proteins; however, some studies have used summed composites of inflammatory markers without first investigating dimensionality. Using two different samples (MIDUS-2: N = 1,255 adults, MIDUS-R: N = 863 adults), this study investigates the dimensionality of eight inflammatory proteins (C-reactive protein (CRP), interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor- (TNF-), fibrinogen, E-selectin, and intercellular adhesion molecule (ICAM)-1) and compared the resulting factor structure to a) an "a priori"/tau-equivalent factor structure in which all inflammatory proteins equally load onto a single dimension (comparable to the summed composites) and b) proteins modeled individually (i.e., no latent variable) in terms of model fit, replicability, reliability, and their associations with health outcomes. An exploratory factor analysis indicated a two-factor structure (Factor 1: CRP and fibrinogen; Factor 2: IL-8 and IL-10) in MIDUS-2 and was replicated in MIDUS-R. Results did not clearly indicate whether the empirically-identified factor structure or the individual proteins modeled without a latent variable had superior model fit, but both strongly outperformed the "a priori"/tau-equivalent structure (which did not achieve acceptable model fit in any models). Modeling the empirically-identified factors and individual proteins (without a latent factor) as outcomes of medical diagnoses resulted in comparable conclusions. However, modeling individual proteins resulted in findings more robust to correction for multiple comparisons despite more conservative adjustments. Further, reliability for all latent variables was poor. These results indicate that modeling inflammation as a unidimensional construct equally associated with all available proteins does not fit the data well. Instead, individual inflammatory proteins or, potentially (if empirically supported and biologically-plausible) empirically-identified inflammatory factors should be used in accordance with theory.

category: immunology


title: A Physiometric Investigation of Inflammatory Composites: Comparison of A Priori Aggregates, Empirically-identified Factors, and Individual Proteins


title: Tau assemblies enter the cytosol of neurons in a cholesterol sensitive manner

abstract:
Accumulating evidence supports a prion-like mechanism in the spread of assembled tau in neurodegenerative diseases. Prion-like spread is proposed to require the transit of tau assemblies to the interior of neurons in order to seed aggregation of native, cytosolic tau. This process is poorly understood and remains largely hypothetical. Here, we develop sensitive techniques to quantify the cytosolic entry of tau in real-time. We find that tau does not promote its own entry but, rather, is wholly dependent on cellular machinery. We find that entry to the widely used reporter cell line HEK293 requires clathrin whereas entry to neurons does not. Cholesterol depletion or knockdown of cholesterol transport protein Niemann-Pick type C1 in neurons renders cells highly vulnerable to cytosolic entry and seeded aggregation. Our findings establish entry as the rate-limiting step in seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation.

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title: Tau assemblies enter the cytosol in a cholesterol sensitive process essential to seeded aggregation


title: High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study

abstract:
summaryWe report insights into genome sequences of Indian K. pneumoniae isolates, highlighting the presence of high-risk international clones and genetic pools different from those predominating in other regions. Identification of multidrug-resistant and hypervirulent K. pneumoniae elicits public health concerns.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India due to its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India.

Materials and MethodsWe characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, MLST, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism (SNP) phylogeny were inferred from their whole genome sequences.

ResultsPhylogenetic analysis of the 325 Klebsiella isolates that passed QC revealed 3 groups: K. pneumoniae sensu stricto (n=307), K. quasipneumoniae (n=17), and K. varicolla (n=1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%), and Col group (35.0%).

ConclusionOur study documents for the first time the genetic diversity of K- and O-antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these virulent and transmissible lineages.

FundingThis work was supported by Official Development Assistance (ODA) funding from the National Institute of Health Research [grant number 16_136_111].

This research was commissioned by the National Institute of Health Research using Official Development Assistance (ODA) funding. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.

Conflict of InterestThe authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.