title: Constrained evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through sequence duplication

abstract:
Neisseria commensals are an indisputable source of resistance for their pathogenic relatives; however, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance ([≤] 2 g/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n=7/16), we find mutations encoding resistance converge on the gene encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through duplication events. Importantly, one of the laboratory evolved mutations in rpmH is identical, and two nearly identical, to those recently reported to confer high-level resistance to azithromycin in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Finally, we assessed the potential of horizontal transfer of derived resistance mutations into multiple strains of N. gonorrhoeae. Though we were unable to transform N. gonorrhoeae in this case, studies like this will be critical for predicting commensal alleles that are at risk of rapid dissemination into pathogen populations.

ImportanceCommensal bacterial populations have been increasingly recognized for their importance as sources of resistance for pathogens, however the collection of antimicrobial resistance (AMR) mechanisms within these communities are often understudied. The risk of reduced antibiotic susceptibility as a result of horizontal gene transfer (HGT) is amplified in highly recombinogenic genera, such as the Neisseria. Indeed, there have been multiple documented cases of macrolide and beta-lactam resistance acquisition in the pathogen N. gonorrhoeae from close commensal relatives. This work uncovers multiple novel azithromycin resistance-conferring mutations in a Neisseria commensal through experimental evolution, investigates their fitness impacts, and explores the possibility of transfer to N. gonorrhoeae. Ultimately these types of studies will illuminate those resistance mutations that may rapidly be acquired across species boundaries.

category: microbiology


title: Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

abstract:
SynopsisO_ST_ABSBackgroundC_ST_ABSThe major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope - a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer - which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb.

Objectives(i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes.

MethodsWe inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal -amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant.

ResultsOur biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, -amylase and polysorbate 80.

ConclusionsSynergistically acting cell wall degrading enzymes are potently inhibiting Mtb - which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.


title: The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress

abstract:
Although ribosome assembly is quality controlled to maintain protein homeostasis, different ribosome populations have been described. How these form, especially under stress conditions that impact energy levels and stop the energy-intensive production of ribosomes, remains unknown. Here we demonstrate how a physiologically relevant ribosome population arises during high Na+ and pH stress via dissociation of Rps26 from fully assembled ribosomes to enable a translational response to these stresses. The chaperone Tsr2 releases Rps26 in the presence of high Na or pH in vitro and is required for Rps26 release in vivo. Moreover, Tsr2 stores free Rps26 and promotes re-incorporation of the protein, thereby repairing the subunit after the stress subsides. Our data implicate a residue in Rps26 involved in Diamond Blackfan Anemia in mediating the effects of Na+. These data demonstrate how different ribosome populations can arise rapidly, without major energy input, and without bypass of quality control mechanisms.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/438496v1_ufig1.gif" ALT="Figure 1">
View larger version (19K):
org.highwire.dtl.DTLVardef@151e234org.highwire.dtl.DTLVardef@d9d3fcorg.highwire.dtl.DTLVardef@ec628corg.highwire.dtl.DTLVardef@13a0917_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LITsr2 releases Rps26 from mature ribosomes to remodel ribosome populations
C_LIO_LITsr2 stores released Rps26 and allows for ribosome repair after stress
C_LIO_LIRps26 exploits a Mg binding site linked to Diamond Blackfan Anemia (DBA)
C_LIO_LIChaperone-mediated ribosome remodeling might be common for chaperoned RPs
C_LI

category: molecular biology


title: Temporal resolution of melanogenesis determine fatty acid metabolism as key skin pigment regulator

abstract: Therapeutic methods to modulate skin pigmentation has important implications for skin cancer prevention and for treating meta-inflammatory-triggered cutaneous conditions. Modulators of cAMP signalling of melanocyte have met with minimal clinical efficacy. Towards defining new potential targets, we followed temporal dynamics orchestrating melanocyte differentiation by using a cell-autonomous pigmentation model. Our study elucidates three dominant phases of synchronized metabolic and transcriptional reprogramming. The induction phase is concomitant with a paradoxical decrease in MITF levels, reduced proliferation, and increased anabolic metabolism mediated by AKT phosphorylation. The melanogenic phase shows rapid uptake of glucose and fatty acid, transiently forming lipid droplets through SREBF1-mediated regulation of fatty acid metabolism. This heightened bioenergetic activity impairs mitochondria and the recovery phase is marked by a shift to aerobic glycolysis and activation of the NRF2 detoxication pathway. Finally, we show that inhibitors of lipid metabolism indeed resolve hyper-pigmentary conditions in a guinea pig UV-tanning model. Our studies reveal metabolic control mechanisms of melanocytes that could govern the balance between differentiation and proliferation in a variety of cutaneous diseases.

category: cell biology


title: Overturning the paradigm that IL6 signaling drives liver regrowth while shining light on a new therapeutic target for regenerative medicine

abstract: It is accepted that IL6 signaling in hepatocytes, mediated via glycoprotein 130 (gp130), is beneficial and that HyperIL6 promotes liver regeneration by activating STAT3. Recently, autocrine IL11 activity, which also signals via gp130 and ERK, was found to be hepatotoxic. Here we examined whether the beneficial effects of HyperIL6 could reflect unappreciated competitive inhibition of IL11 signaling. In hepatocytes, HyperIL6 inhibited N-acetyl-p-aminophenol (APAP)-induced cell death that mimicked inhibition of IL11 signaling and was unrelated to STAT3 phosphorylation. In mice, expression of HyperIL6 reduced liver damage due to IL11 dosing or APAP and promoted hepatic regeneration in a STAT3-independent manner. Following APAP, mice deleted for Il11 were protected from liver failure and exhibited spontaneous regeneration. Despite robustly activating STAT3, HyperIL6 had no beneficial effect in Il11 null mice. These data overturn the premise that IL6 promotes liver regeneration, show STAT3 activation to be redundant and suggest IL11 as a focus for regenerative medicine.


title: R-SPONDIN2+ Mesenchymal Cells Form the Bud Tip Progenitor Niche During Human Lung Development

abstract: Mammalian respiratory system development is regulated by complex reciprocal signaling events that take place between epithelial cells and the surrounding mesenchymal cells; however, mesenchymal heterogeneity and function in the developing human lung is poorly understood. We interrogated single cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist R-SPONDIN2 (RSPO2), and we found that adjacent epithelial bud tip progenitors are enriched for the RSPO2 receptor LGR5. By carrying out functional experiments using organoid models, lung explant cultures, and FACS-isolated RSPO2+ mesenchyme, we show that RSPO2 is a critical niche cue that potentiates WNT signaling in human lung progenitors to maintain their multipotency.

category: developmental biology


title: East-African savanna dynamics: from a knowledge-based model to the possible futures of a social-ecological system

abstract: Sub-Saharan savanna ecosystems are undergoing transitions such as bush encroachment, desertification or agricultural expansion. Such shifts and persistence of land cover are increasingly well understood, especially bush encroachment which is of major concern in pastoral systems. Although dominant factors can explain such transformations, they often result from intertwined causes in which human activities play a significant role. Therefore, in this latter case, these issues may require integrated solutions, involving many interacting components. Ecosystem modelling has proved appropriate to support decision-makers in such complex situations. However, ecosystem models often require lots of quantitative information for estimating parameters and the precise functional form of interactions is often unknown. Alternatively, in rangeland management, States-and-Transitions Models (STMs) have been developed to organize knowledge about system transitions and to help decision-makers. However, these conceptual diagrams often lack mathematical analyzing tools, which strongly constrains their complexity. In this paper, we introduce the Ecological Discrete-Event Network (EDEN) modelling approach for representing the qualitative dynamics of an East-African savanna as a set of discrete states and transitions generated from empirical rules. These rules are derived from local knowledge, field observations and scientific literature. In contrast with STMs, EDEN generates automatically every possible states and transitions, thus enabling the prediction of novel ecosystem structures. Our results show that the savanna is potentially resilient to the disturbances considered. Moreover, the model highlights all transitions between vegetation types and socio-economic profiles under various climatic scenarios. The model also suggests that wildlife diversity may increase socio-economic resistance to seasonal drought. Tree-grass coexistence and agropastoralism have the widest ranges of conditions of existence of all vegetation types and socio-economic profiles, respectively. As this is a preliminary use of EDEN for applied purpose, analysis tools should be improved to enable finer investigation of desirable trajectories. By translating local knowledge into ecosystem dynamics, the EDEN approach seems promising to build a new bridge between managers and modellers.

category: ecology


title: Regulation of DIM-2-dependent repeat-induced point mutation (RIP) by the recombination-independent homologous DNA pairing in Neurospora crassa

abstract:
Repeat-induced point mutation (RIP) is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. RIP detects duplications irrespective of their origin, particular sequence, coding capacity, or genomic positions. Previous studies suggested that RIP involves a cardinally new mechanism of sequence recognition that operates on intact double-stranded DNAs. In the fungus Neurospora crassa, RIP can be mediated by a putative C5-cytosine methyltransferase (CMT) RID or/and a canonical CMT DIM-2. These distinct RIP pathways feature opposite substrate preferences: RID-dependent RIP is largely limited to the duplicated sequences, whereas DIM-2-dependent RIP preferentially mutates adjacent non-repetitive regions. Using DIM-2-dependent RIP as a principal readout of repeat recognition, here we show that GC-rich repeats promote stronger RIP compared to AT-rich repeats (independently of their intrinsic propensities to become mutated), with the relative contribution of AT base-pairs being close to zero. We also show that direct repeats promote much more efficient DIM-2-dependent RIP than inverted repeats; both the spacer DNA between the repeat units (the linker) and the flanking regions are similarly affected by this process. These and other results support the idea that repeat recognition for RIP involves formation of many short interspersed quadruplexes between homologous double-stranded DNAs, which need to undergo concomitant changes in their linking number to accommodate pairing.

SUMMARYDuring repeat-induced point mutation (RIP) gene-sized duplications of genomic DNA are detected by a mechanism that likely involves direct pairing of homologous double-stranded DNAs. We show that DIM-2-dependent RIP, triggered by closely-positioned duplications, is strongly affected by their relative orientations (direct versus inverted). We also show that GC-rich repeats promote RIP more effectively than AT-rich repeats. These results support a model in which homologous dsDNAs can pair by establishing interspersed quadruplex-based contacts with concomitant changes in their supercoiling status.

category: genetics


title: Palmerolide PKS-NRPS gene clusters characterized from the microbiome of an Antarctic ascidian

abstract: Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the [~]75 kb trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicohabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

category: biochemistry


title: Bioinformatic and mechanistic analysis of the palmerolide PKS-NRPS biosynthetic pathway from the microbiome of an Antarctic ascidian


title: Genetic Encoding of a Highly Photostable, Long Lifetime Fluorescent Amino Acid for Imaging in Mammalian Cells

abstract: Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.


title: OGUs enable effective, phylogeny-aware analysis of even shallow metagenome community structures

abstract:
We introduce Operational Genomic Unit (OGU), a metagenome analysis strategy that directly exploits sequence alignment hits to individual reference genomes as the minimum unit for assessing the diversity of microbial communities and their relevance to environmental factors. This approach is independent from taxonomic classification, granting the possibility of maximal resolution of community composition, and organizes features into an accurate hierarchy using a phylogenomic tree. The outputs are suitable for contemporary analytical protocols for community ecology, differential abundance and supervised learning while supporting phylogenetic methods, such as UniFrac and phylofactorization, that are seldomly applied to shotgun metagenomics despite being prevalent in 16S rRNA gene amplicon studies. As demonstrated in one synthetic and two real-world case studies, the OGU method produces biologically meaningful patterns from microbiome datasets. Such patterns further remain detectable at very low metagenomic sequencing depths. Compared with taxonomic unit-based analyses implemented in currently adopted metagenomics tools, and the analysis of 16S rRNA gene amplicon sequence variants, this method shows superiority in informing biologically relevant insights, including stronger correlation with body environment and host sex on the Human Microbiome Project dataset, and more accurate prediction of human age by the gut microbiomes in the Finnish population. We provide Woltka, a bioinformatics tool to implement this method, with full integration with the QIIME 2 package and the Qiita web platform, to facilitate OGU adoption in future metagenomics studies.

ImportanceShotgun metagenomics is a powerful, yet computationally challenging, technique compared to 16S rRNA gene amplicon sequencing for decoding the composition and structure of microbial communities. However, current analyses of metagenomic data are primarily based on taxonomic classification, which is limited in feature resolution compared to 16S rRNA amplicon sequence variant analysis. To solve these challenges, we introduce Operational Genomic Units (OGUs), which are the individual reference genomes derived from sequence alignment results, without further assigning them taxonomy. The OGU method advances current read-based metagenomics in two dimensions: (i) providing maximal resolution of community composition while (ii) permitting use of phylogeny-aware tools. Our analysis of real-world datasets shows several advantages over currently adopted metagenomic analysis methods and the finest-grained 16S rRNA analysis methods in predicting biological traits. We thus propose the adoption of OGU as standard practice in metagenomic studies.

category: bioinformatics


title: Identification of biomarkers and pathways of mouse embryonic fibroblasts with the dysfunction of mitochondrial DNA

abstract: Mitochondrial diseases are clinically heterogeneous which involve multiple systems such as organs that are highly dependent on metabolism. Dysfunction of mtDNA is the main cause of mitochondrial diseases that trigger inflammation and immune responses. Here, we aim to identify the biological function and pathways of MEFs with the dysfunction of mtDNA through deletion of YME1L. The gene expression profiles of GSE161735 dataset were originally created by the Illumina NovaSeq 6000 (Mus musculus) for gene biogenesis and function panel. The biological and functional pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Gene Ontology (GO), and Reactom visual map. KEGG and GO results showed the metabolism and immune responses were mostly affected by the loss of mtDNA. Moreover, we discovered several interacting genes including POLR2F, HIST1H2BJ, PPP1CC, HOXB4, ARG1, APITD1, BUB1B, POLR2K, HOXC4, and HOXB3 were involved in the regulation of metabolic or cancer diseases. Further, we predicted several regulators that had the ability to affect mitochondria during the dysfunction of mtDNA by L1000fwd analysis. Thus, this study provides further insights into the mechanism of mtDNA in metabolic diseases.


title: Suppression of endothelial miR-22-3p mediates non-small cell lung cancer cell-induced angiogenesis

abstract: MicroRNAs (miRNAs) expressed in endothelial cells (ECs) are powerful regulators of angiogenesis, which is essential for tumor growth and metastasis. Here, we demonstrated that miR-22-3p (miR-22) is preferentially and highly expressed in ECs, while its endothelial level is significantly down-regulated in human non-small cell lung cancer (NSCLC) tissues when compared to matched non-tumor lung tissues. This reduction of endothelial miR-22 is induced by NSCLC cell-secreted tumor necrosis factor (TNF)- and interleukin (IL)-1{beta}. Endothelial miR-22 functions as a potent angiogenesis inhibitor that inhibits all the key angiogenic activities of ECs and consequently NSCLC growth through directly targeting sirtuin (SIRT) 1 and fibroblast growth factor receptor (FGFR) 1 in ECs, leading to inactivation of AKT/mammalian target of rapamycin (mTOR) signaling. These novel findings provide insight into the molecular mechanisms of NSCLC angiogenesis and indicate that endothelial miR-22 represents a potential target for the future anti-angiogenic treatment of NSCLC.

category: cancer biology


title: Warped Bayesian Linear Regression for Normative Modelling of Big Data

abstract:
Normative modelling is becoming more popular in neuroimaging due to its ability to make predictions of deviation from a normal trajectory at the level of individual participants. It allows the user to model the distribution of several neuroimaging modalities, giving an estimation for the mean and centiles of variation. With the increase in the availability of big data in neuroimaging, there is a need to scale normative modelling to big data sets. However, the scaling of normative models has come with several challenges.

So far, most normative modelling approaches used Gaussian process regression, and although suitable for smaller datasets (up to a few thousand participants) it does not scale well to the large cohorts currently available and being acquired. Furthermore, most neuroimaging modelling methods that are available assume the predictive distribution to be Gaussian in shape. However, deviations from Gaussianity can be frequently found, which may lead to incorrect inferences, particularly in the outer centiles of the distribution. In normative modelling, we use the centiles to give an estimation of the deviation of a particular participant from the  normal trend. Therefore, especially in normative modelling, the correct estimation of the outer centiles is of utmost importance, which is also where data are sparsest.

Here, we present a novel framework based on Bayesian Linear Regression with likelihood warping that allows us to address these problems, that is, to scale normative modelling elegantly to big data cohorts and to correctly model non-Gaussian predictive distributions. In addition, this method provides also likelihood-based statistics, which are useful for model selection.

To evaluate this framework, we use a range of neuroimaging-derived measures from the UK Biobank study, including image-derived phenotypes (IDPs) and whole-brain voxel-wise measures derived from diffusion tensor imaging. We show good computational scaling and improved accuracy of the warped BLR for certain IDPs and voxels if there was a deviation from normality of these parameters in their residuals.

The present results indicate the advantage of a warped BLR in terms of; computational scalability and the flexibility to incorporate non-linearity and non-Gaussianity of the data, giving a wider range of neuroimaging datasets that can be correctly modelled.

category: neuroscience


title: An Autoantigen-ome from HS-Sultan B-Lymphoblasts Offers a Molecular Map for Investigating Autoimmune Sequelae of COVID-19

abstract: To understand how COVID-19 may induce autoimmune diseases, we have been compiling an atlas of COVID-autoantigens (autoAgs). Using dermatan sulfate (DS) affinity enrichment of autoantigenic proteins extracted from HS-Sultan lymphoblasts, we identified 362 DS-affinity proteins, of which at least 201 (56%) are confirmed autoAgs. Comparison with available multi-omic COVID data shows that 315 (87%) of the 362 proteins are affected in SARS-CoV-2 infection via altered expression, interaction with viral components, or modification by phosphorylation or ubiquitination, at least 186 (59%) of which are known autoAgs. These proteins are associated with gene expression, mRNA processing, mRNA splicing, translation, protein folding, vesicles, and chromosome organization. Numerous nuclear autoAgs were identified, including both classical ANAs and ENAs of systemic autoimmune diseases and unique autoAgs involved in the DNA replication fork, mitotic cell cycle, or telomerase maintenance. We also identified many uncommon autoAgs involved in nucleic acid and peptide biosynthesis and nucleocytoplasmic transport, such as aminoacyl-tRNA synthetases. In addition, this study found autoAgs that potentially interact with multiple SARS-CoV-2 Nsp and Orf components, including CCT/TriC chaperonin, insulin degrading enzyme, platelet-activating factor acetylhydrolase, and the ezrin-moesin-radixin family. Furthermore, B-cell-specific IgM-associated ER complex (including MBZ1, BiP, heat shock proteins, and protein disulfide-isomerases) is enriched by DS-affinity and up-regulated in B-cells of COVID-19 patients, and a similar IgH-associated ER complex was also identified in autoreactive pre-B1 cells in our previous study, which suggests a role of autoreactive B1 cells in COVID-19 that merits further investigation. In summary, this study demonstrates that virally infected cells are characterized by alterations of proteins with propensity to become autoAgs, thereby providing a possible explanation for infection-induced autoimmunity. The COVID autoantigen-ome provides a valuable molecular resource and map for investigation of COVID-related autoimmune sequelae and considerations for vaccine design.

category: immunology


title: Comparative analyses of gene expression in common marmoset and human pluripotent stem cells (PSCs) identify factors enhancing homologous recombination efficiency in the HPRT locus of human PSCs

abstract: A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination (HR) activity in marmoset ESCs (cmESCs). Here, we compared gene expression in marmoset and human pluripotent stem cells (PSCs) using transcriptomic and quantitative PCR (qPCR) analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D and RAD51) were upregulated in marmoset cells. Four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.


title: Modeling the role of mortality-based response triggers on the effectiveness of African swine fever control strategies

abstract:
African swine fever (ASF) is considered the most impactful transboundary swine disease. In the absence of effective vaccines, control strategies are heavily dependent on mass depopulation and movement restrictions. Here we developed a nested multiscale model for the transmission of ASF, combining spatially explicit network model of animal movements with a deterministic compartmental model for the dynamics of two ASF strains within-pixels of 3 km x 3 km, amongst the pig population in one Brazilian state. The model outcomes are epidemic duration, number of secondary infected farms and pigs, and distance of ASF spread. The model also shows the spatial distribution of ASF epidemics. We analyzed quarantine-based control interventions in the context of mortality trigger thresholds for the deployment of control strategies.

The mean epidemic duration of a moderately virulent strain was 11.2 days assuming the first infection is detected (best-case scenario) and 15.9 days when detection is triggered at 10 % mortality. For a highly virulent strain, the epidemic duration was 6.5 days and 13.1 days, respectively. The distance from the source to infected locations and the spatial distribution was not dependent on strain virulence. Under the best-case scenario, we projected an average number of infected farms of 23.77 farms and 18.8 farms for the moderate and highly virulent strains, respectively. At 10% mortality-trigger, the predicted number of infected farms was on average 46.27 farms and 42.96 farms, respectively. We also demonstrated that the establishment of ring quarantine zones regardless of size (i.e., 5 km, 15 km) was outperformed by backward animal movement tracking. The proposed modeling framework provides an evaluation of ASF epidemic potential, providing a ranking of quarantine-based control strategies that could assist animal health authorities in planning the national preparedness and response plan.


title: Heatrich-BS enables efficient CpG enrichment and highly scalable cell-free DNA methylation profiling

abstract: Genome wide analysis of cell-free DNA (cfDNA) methylation profile has been shown to be a promising approach for sensitive and specific multi-cancer detection. However, scaling these assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We showed that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbored the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that enables focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable high accuracy and sensitivity in tumor burden estimation and quantitative monitoring of colorectal patient response to treatment, at much reduced sequencing requirement. Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

category: genomics


title: Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi

abstract: Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic sequence variation in the ITS2-rDNA of An. stephensi and hypothesizes that observed intra-specific differences in this species may have resulted due to ambiguous DNA sequence-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S-rDNA (d1-d3 domains). Intragenomic variations were found in ITS2 region of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S-rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central, and southern India confirmed the presence of intragenomic variation in ITS2 due to transitions at three loci and two bp indel in a di-nucleotide microsatellite locus. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.


title: Novel human cell expression method reveals the role and prevalence of posttranslational modification in non-muscle tropomyosins

abstract: Biochemical studies require large protein quantities, which are typically obtained using bacterial expression. However, the folding machinery of bacteria is inadequate for many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for function. Tropomyosin, a coiled coil that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation, to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several non-muscle tropomyosin isoforms. Mammalian cell-expressed tropomyosins are functionally different from their E. coli-expressed counterparts, display multiple types of PTMs, and can form heterodimers. This method can be extended to other proteins, as demonstrated here for -synuclein.


title: HandyCNV: Standardized Summary, Annotation, Comparison, and Visualization of CNV, CNVR and ROH

abstract:
Here we present an R package for summarizing, annotating, converting, comparing and visualizing CNV (copy number variants) and ROH (runs of homozygosity) detected from SNP (single nucleotide polymorphism) genotyping data. This one-stop post-analysis system is standardized, comprehensive, reproducible, timesaving and user friendly for research in humans and most diploid livestock species.

Availability and ImplementationThe source code, demo data and vignettes can be found at https://github.com/JH-Zhou/HandyCNV


title: DeepTrio: Variant Calling in Families Using Deep Learning

abstract: Every human inherits one copy of the genome from their mother and another from their father. Parental inheritance helps us understand the transmission of traits and genetic diseases, which often involve de novo variants and rare recessive alleles. Here we present DeepTrio, which learns to analyze child-mother-father trios from the joint sequence information, without explicit encoding of inheritance priors. DeepTrio learns how to weigh sequencing error, mapping error, and de novo rates and genome context directly from the sequence data. DeepTrio has higher accuracy on both Illumina and PacBio HiFi data when compared to DeepVariant. Improvements are especially pronounced at lower coverages (with 20x DeepTrio roughly equivalent to 30x DeepVariant). As DeepTrio learns directly from data, we also demonstrate extensions to exome calling solely by changing the training data. DeepTrio includes pre-trained models for Illumina WGS, Illumina exome, and PacBio HiFi.


title: An intein-split transactivator for intersectional neural imaging and optogenetic manipulation

abstract: The complexity of brain circuitry is manifested by numerous cell types based on genetic marker, location and neural connectivity. Cell-type specific recording and manipulation is essential to disentangle causal neural mechanisms in physiology and behavior; however, many current approaches are largely limited by number of intersectional features, incompatibility of common effectors and insufficient gene expression. To tackle these limitations, we devise an intein-based intersectional synthesis of transactivator (IBIST) to selectively control gene expression of common effectors in specific cell types defined by a combination of multiple features. We validate the specificity and sufficiency of IBIST to control common effectors including fluorophores, optogenetic opsins and Ca2+ indicators in various intersectional conditions in vivo. Using IBIST-based Ca2+ imaging, we show that the IBIST can intersect up to five features, and that hippocampal cells tune differently to distinct emotional valences depending on the pattern of projection targets. Collectively, the IBIST multiplexes the capability to intersect cell-type features and is compatible with common effectors to effectively control gene expression, monitor and manipulate neural activities.


title: Feedback-related potentials and oscillations during trial and error learning in Parkinson's disease

abstract:
Electrophysiological markers of performance monitoring are thought to reflect functioning of dedicated neural networks and neuromodulatory systems. Whether and how these markers are altered in neurological diseases and whether they can reflect particular cognitive deficits remains to be confirmed. Here we first tested whether the frontal medial feedback-related potential, evoked during a trial and error learning task, is changed in early Parkinsons disease patients compared to control subjects. The potential was not changed in amplitude and discriminated negative and positive feedback as in controls. Feedback-related markers in Parkinsons patients also appeared in time-frequency analyses, unaltered in theta (3-7 Hz) band but reduced in beta (20-30 Hz) oscillations for positive feedback. Beta oscillations power appeared to be dramatically globally reduced during the task. Overall, our results show that Beta oscillation markers of performance monitoring captured by EEG are selectively altered in Parkinsons disease patients, and that they are accompanied by changes in task-related oscillatory dynamics.

Significance StatementFrontal neural activity evoked by outcomes reveal the functioning of neural systems devoted to flexible behaviours. Modulations of such activity in Parkinsons disease (PD) patients could reflect specific alterations of neural systems and cognitive processing. The goal is to evaluate whether such activity can serve as markers of the disease. Here, using EEG and a trial and error learning protocol, we show that mid-frontal midline performance feedback-related potentials for different types of feedback were similar in controls and early diagnosed PD patients. However, task-related oscillations revealed alterations in the beta range accompanied by more global beta activity alteration in PD compared to controls subjects. This study provides data relevant to the search for non-motor biomarkers in early stages of PD.


title: Soil, ocean, hot spring, and host-associated environments reveal unique selection pressures on genomic features of bacteria in microbial communities

abstract: Free-living bacteria in nutrient limited environments often exhibit small genomes which curb the cost of reproduction - a phenomenon known as genomic streamlining. Streamlining has been associated with a suite of traits such as reduced GC content, fewer 16S rRNA copies, and a lower abundance of regulatory genes, such as sigma ({sigma}) factors. Here, we analyzed these traits from 116 publicly available metagenomes derived from marine, soil, host associated, and thermophilic communities. In marine and thermophilic communities, genome size and GC content declined in parallel, but GC content was higher in thermophilic communities. In soils, the relationship between genome size and GC content was negative, suggesting a different selection pressure on genome size and GC content in soil bacteria. The abundance of {sigma}-factors varied with average genome size, ecosystem type, and the specific functions regulated by the sigma factor. In marine environments, housekeeping and heat-shock {sigma}-factor genes (rpoD and rpoH respectively) increased as genome size declined, and {sigma}-factor responsible for flagella biosynthesis (fliA) decreased, suggesting a trade-off between nutrient conservation and chemotaxis. In soils, a high abundance of fliA and the stress response {sigma}-factor gene (rpoS) was associated with smaller average genome size and often located in harsh and/or carbon-limited environments such as deserts or agricultural fields - suggesting an increased capacity for stress response and mobility in nutrient-poor soils. This work showcases how ecosystem-specific environmental constraints force trade-offs which are then embedded in the genomic features of bacteria in microbial communities, specifically genome size, GC content, and regulatory genes, and further highlights the importance of considering these features in microbial community analysis.


title: Introducing Conflict Resolution and Negotiation Training into a Biomedical Sciences Graduate Curriculum

abstract: Analysis of the biomedical workforce and graduate education have produced recommendations for modifications of pre-doctoral training to broadly prepare trainees for wider ranging scientific careers. Increased exposure to career opportunities and development of training in professional skills is widely recommended, but details of implementation are not widely available. In alignment with these recommendations, we have incorporated professional skills training into the biomedical science graduate curriculum at West Virginia University. An important component of the training is developing conflict resolution and negotiation skills. This training will provide useful skills for academic careers, non-academic careers and life situations outside of the workplace. Conflict resolution/negotiation skills are also relevant in managing issues in diversity, equity and inclusivity. We report our experience in developing this component of the training program, provide an overview of the approach to delivery and practice of skills, and provide an analysis of the reception and effectiveness of the training.

category: scientific communication and education


title: Large-Scale Deep Multi-Layer Analysis of Alzheimer's Disease Brain Reveals Strong Proteomic Disease-Related Changes Not Observed at the RNA Level

abstract: The biological processes that are disrupted in the Alzheimers disease (AD) brain remain incompletely understood. We recently performed a proteomic analysis of >2000 brains to better understand these changes, which highlighted alterations in astrocytes and microglia as likely key drivers of disease. Here, we extend this analysis by analyzing >1000 brain tissues using a tandem mass tag mass spectrometry (TMT-MS) pipeline, which allowed us to nearly triple the number of quantified proteins across cases. A consensus protein co-expression network analysis of this deeper dataset revealed new co-expression modules that were highly preserved across cohorts and brain regions, and strongly altered in AD. Nearly half of the protein co-expression modules, including modules significantly altered in AD, were not observed in RNA networks from the same cohorts and brain regions, highlighting the proteopathic nature of AD. Two such AD-associated modules unique to the proteomic network included a module related to MAPK signaling and metabolism, and a module related to the matrisome. Analysis of paired genomic and proteomic data within subjects showed that expression level of the matrisome module was influenced by the APOE {varepsilon}4 allele, but was not related to the rate of cognitive decline after adjustment for neuropathology. In contrast, the MAPK/metabolism module was strongly associated with the rate of cognitive decline. Disease-associated modules unique to the proteome are sources of promising therapeutic targets and biomarkers for AD.

category: systems biology


title: Large-scale voltage imaging in the brain using targeted illumination

abstract: Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold membrane voltage dynamics from single neurons in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool for recording activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy more prone to background signal contamination, and thus far voltage imaging using fully genetically encoded voltage indicators remains limited to simultaneous sampling of a few cells over a restricted field-of-view. We here demonstrate a strategy for large scale voltage imaging using the fully genetically encoded voltage indicator SomArchon and targeted illumination. We implemented a simple, low-cost digital micromirror device based targeted illumination strategy to restrict illumination to the cells of interest, and systematically quantified the improvement of this microscopy design theoretically and experimentally with SomArchon expressing neurons in single layer cell cultures and in the brains of awake mice. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brain. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed, large area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in the brain. Thus, the widefield microscopy design with an integrated targeted illumination system described here offers a simple solution for voltage imaging analysis of large neuron populations in behaving animals.


title: Selective remembering: Cue overlap enables pre-retrieval control of recollection

abstract: People often want to recall only currently relevant events, but this selective remembering is not always possible. We contrasted two candidate mechanisms: the overlap between retrieval cues and stored memory traces, and the ease of recollection. In two preregistered experiments (Ns = 28) we used event-related potentials (ERPs) to quantify pre-retrieval selection and the goal states - retrieval orientations - thought to achieve this selection. Participants viewed object pictures or heard object names, and one of these sources was designated as targets in each memory test. We manipulated cue overlap by probing memory with visual names (Experiment 1) or line drawings (Experiment 2). Results revealed that regardless of which source was targeted, the left parietal ERP effect indexing recollection was selective when test cues overlapped more with the targeted than non-targeted information, despite consistently better memory for pictures. ERPs for unstudied items were also more positive-going when cue overlap was high, suggesting that engagement of retrieval orientations reflected availability of external cues matching the targeted source. The data support the view that selection can act prior to recollection if there is sufficient overlap between retrieval cues and targeted versus competing memory traces.


title: Cue overlap supports pre-retrieval selection in episodic memory: ERP evidence


title: Single-cell sequencing unveils distinct immune microenvironment with CCR6-CCL20 crosstalk in human chronic pancreatitis

abstract:
ObjectiveChronic pancreatitis (CP) is a potentially fatal disease of the exocrine pancreas, with no specific or effective approved therapies. Due to difficulty in accessing pancreas tissues, little is known about local immune responses or pathogenesis in human CP. We sought to characterize pancreas immune responses using tissues derived from patients with different etiologies of CP and non-CP organ donors in order to identify key signaling molecules associated with human CP.

DesignWe performed single-cell level cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) and T cell receptor sequencing of pancreatic immune cells isolated from organ donors, hereditary, and idiopathic CP patients who underwent total pancreatectomy. We validated gene expression data by performing flow cytometry and functional assay in the second CP patient cohort.

ResultsDeep single-cell sequencing revealed distinct immune characteristics and significantly enriched CCR6+ CD4+ T cells in hereditary compared with idiopathic CP. In hereditary CP, a reduction in T cell clonality was observed due to the increased CD4+ T (Th) cells that replaced tissue-resident CD8+ T cells. Shared TCR clonotype analysis among T cell lineages also unveiled unique interactions between CCR6+ Th and Th1 subsets, and TCR clustering analysis showed unique common antigen binding motifs in hereditary CP. In addition, we observed a significant upregulation of the CCR6 ligand (CCL20) among monocytes in hereditary CP as compared with those in idiopathic CP. The functional significance of CCR6 expression in CD4+ T cells was confirmed by flow cytometry and chemotaxis assay.

ConclusionSingle-cell sequencing with pancreatic immune cells in human CP highlights pancreas-specific immune crosstalk through the CCR6-CCL20 axis that might be leveraged as a potential future target in human hereditary CP.

Significance of this studyO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIChronic pancreatitis (CP) is considered an irreversible fibroinflammatory pancreatic disease and remains a major source of morbidity among gastrointestinal diseases with no active approved therapy.
C_LIO_LIInflammation is a known hallmark and contributor to CP pathogenesis. However, little is known about local immune responses in human CP especially with different etiologies.
C_LI

What are the new findings?O_LISingle-cell RNA sequencing of pancreatic immune cells from CP patients and organ donors revealed distinct immune transcriptomic features in CP versus non-diseased controls and hereditary versus idiopathic CP.
C_LIO_LISingle-cell T cell receptor sequencing unveiled pancreas-specific clonal expansion in CD8+ T cells and CD4+ T cells-driven unique TCR repertoire changes in hereditary CP.
C_LIO_LIWe identified that the CCR6-CCL20 axis was significantly upregulated in hereditary CP compared with controls or idiopathic CP suggesting a potential future target for human hereditary CP.
C_LI

How might it impact on clinical practice in the foreseeable future?O_LIThe results of this study will improve our understanding of CP heterogeneity and identify distinct immune responses in different types of human CP that could provide novel conceptual directions for therapeutic strategies in treating hereditary and idiopathic CP.
C_LIO_LIThe CCR6-CCL20 axis found in hereditary CP could be a potential novel targetable signaling pathways in the treatment of hereditary CP.
C_LI


title: A novel perspective on the meaning of nestedness with conceptual and methodological solutions

abstract:
O_LINestedness is a common pattern in metacommunities and interaction networks, whose causes are still subject to investigation. Nestedness inference is challenging because, beyond calculating an index, we need to compare observed and expected values using null models. There are many different null models and the choice between them strongly affects test outcomes. Furthermore, there is no theoretical basis to guide this choice.
C_LIO_LIHere, we propose a different look at the meaning of nestedness that improves our understanding of its causes and directly links null models to hypotheses. The nestedness of a matrix is the combination of marginal sum inequality and high overlap. The higher the overlap, the more predictable the cell values by marginal sums. Therefore, the only way a process can increase nestedness is by promoting marginal sum inequalities, without concomitantly introducing preferences. We propose a combination of null models to test for different topological hypotheses. In these null models, interactions are randomized following a probability matrix. The probability matrix of the equiprobable model excludes marginal sum inequalities and consequently all nestedness-generating mechanisms, providing a distribution of expected values for nestedness significance tests. The probability matrix of the proportional model, by conserving marginal sums and maximizing overlap, conserves nestedness-generating mechanisms and excludes nestedness-disrupting mechanisms, thereby yielding the expected nestedness values for fully nested matrices.
C_LIO_LIWe advance a new protocol to test for nestedness in binary and weighted matrices, in which those two null models represent different topological hypotheses. Our protocol helps to tell apart cases in which nestedness is different from expected by chance and cases in which nestedness represents the best topological archetype. We also evaluate the efficiency of applying this protocol using some common nestedness indices. Analyses based on weighted data proved to be highly efficient and informative, while binary analyses induced severe distortions. Finally, we empirically illustrate our approach using a plant-pollinator network.
C_LIO_LIThrough a shift of perspective, our approach reconciliates contradictions in null model analysis and provides a guideline for nestedness interpretation.
C_LI


title: Linking null models to hypotheses to improve nestedness analysis


title: Brassica rapa domestication: untangling wild and feral forms and convergence of crop morphotypes

abstract: The study of domestication contributes to our knowledge of evolution and crop genetic resources. Human selection has shaped wild Brassica rapa into diverse turnip, leafy, and oilseed crops. Despite its worldwide economic importance and potential as a model for understanding diversification under domestication, insights into the number of domestication events and initial crop(s) domesticated in B. rapa have been limited due to a lack of clarity about the wild or feral status of conspecific non-crop relatives. To address this gap and reconstruct the domestication history of B. rapa, we analyzed 68,468 genotyping-by-sequencing-derived SNPs for 416 samples in the largest diversity panel of domesticated and weedy B. rapa to date. To further understand the center of origin, we modeled the potential range of wild B. rapa during the mid-Holocene. Our analyses of genetic diversity across B. rapa morphotypes suggest that non-crop samples from the Caucasus, Siberia, and Italy may be truly wild, while those occurring in the Americas and much of Europe are feral. Clustering, tree-based analyses, and parameterized demographic inference further indicate that turnips were likely the first crop type domesticated, from which leafy types in East Asia and Europe were selected from distinct lineages. These findings clarify the domestication history and nature of wild crop genetic resources for B. rapa, which provides the first step toward investigating cases of possible parallel selection, the domestication and feralization syndrome, and novel germplasm for Brassica crop improvement.

category: evolutionary biology


title: Inversions maintain differences between migratory phenotypes of a songbird

abstract: Long-distance migration requires adaptations in a suite of behavioral, physiological and morphological traits. However, almost nothing is known about the genetic basis of these adaptations. The willow warbler Phylloscopus trochilus occurs in Europe with a northern and southern subspecies that show drastically different migration routes and wintering areas. Previous studies have demonstrated that the subspecies are genetically extremely similar except for three divergent chromosome regions, of which two are associated with the differences in migratory phenotypes and one is associated with an environmental gradient. Here we use a combination of long-read sequencing, linked-read sequencing and optical mapping to construct more complete and contiguous assemblies for both of the subspecies. We find evidence for inversions in each of the three divergent regions, which range from 0.4 to 13 Mb in size, and that breakpoints are associated with tandem repeat arrays or segmental duplications. The divergence times between inverted and non-inverted haplotypes are similar across the regions ([~]1.2 Myrs), which is compatible with a scenario where the inversions arose in either of two allopatric populations that subsequently hybridized. The improved genome assemblies and annotation also allowed us to detect additional functional differences in the divergent regions that provide candidate genes for migration and local adaptations to environmental gradients.


title: Multi-omics approach identifies germline regulatory variants associated with hematopoietic malignancies in retriever dog breeds

abstract: Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (Pwald=4.83x10-9) and 19 (Pwald=2.25x10-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility.


title: Connection of core and tail Mediator modules restrains transcription from TFIID-dependent promoters

abstract: The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels of PIC formation at SAGA-requiring and TFIID-dependent genes. We suggest that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID.


title: Optimizing touchscreen measures of rodent cognition by eliminating image bias

abstract: For the last twenty years, the Bussey-Saksida touchscreen-based operant conditioning platform has evolved in close parallel alongside the Cambridge Neuropsychological Test Automated Battery (CANTAB) to produce batteries of tests for studying complex cognitive functions in rodents that are increasingly analogous to human diagnostic tests and greatly narrow the translational gap in cognition research. Naturally, with this increasing usefulness comes increasing use, particularly by non-experts. This necessitates a greater understanding of, and a better controlling for, confounding factors that may limit the systems ability to optimally detect cognitive deficits when used as a widely accessible and commercially available standardized task. In the present study, we show a strong image preference bias in a standard pairwise discrimination task with a widely used spider-plane image pairing in a putative animal model for intellectual disability. This bias greatly influenced the performance of our experimental mice, significantly affecting the length of time it took mice to complete the task, their progress over time, and several accessory measures usefully recorded by the Bussey-Saksida touchscreen system. We further show that this bias can be corrected by using more similar image pairings without sacrificing the animals ability to learn to distinguish the stimuli. This approach eliminated all significant stimuli specific differences seen with the spider-plane pairing. We then analyzed the pixel composition of the various stimuli to suggest that the bias is due to a difference in image brightness. These findings highlight the importance of carefully modulating paired touchscreen stimuli to ensure equivalence prior to learning and the need for more studies of visual perception in mice, particularly as it relates to their performance in cognitive assays.

category: animal behavior and cognition


title: Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity

abstract:
Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for corynespora leaf fall disease in rubber trees. Currently, the molecular mechanism of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane-disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more susceptible to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Cytotoxicity tests on rubber leaves of RRIV 1, RRIV 4, and PB 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryo- SEM analyses of necrotic leaf tissues exposed to Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and disrupt specific membranes.

Graphic Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/438527v2_ufig1.gif" ALT="Figure 1">
View larger version (42K):
org.highwire.dtl.DTLVardef@17ef3f4org.highwire.dtl.DTLVardef@1c24d41org.highwire.dtl.DTLVardef@15e071aorg.highwire.dtl.DTLVardef@24740e_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LICas1 and Cas2 directly damage and cause necrosis in the leaves of specific rubber clones.
C_LIO_LICas1 and Cas2 can form biofilm-like structures on specific lipid membranes (negative phospholipids, glycerolipids, and sterols). The biofilm-like formation of Cas toxin plays an important role in selective disruption on lipid membranes.
C_LIO_LIVulnerability of the specific cytoplasmic membranes to the selective Cas toxin that is the most remarkable feature of cytotoxicity of Cas toxin on plant cells.
C_LI


category: pathology


title: A CRISPR/Cas12a-assisted platform for identification and quantification of single CpG methylation sites

abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) systems have repeatedly shown to have excellent performance in nucleotide sensing applications1-5. High specificity and selectivity of Cas effector proteins is determined by the CRISPR RNAs (crRNAs) interchangeable spacer sequence, as well as position and number of mismatches between target sequence and the crRNA sequence1. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate a method to discriminate single CpG site methylation in DNA, which is an epigenetic alteration, by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene were for the first time determined. The modularity of both Cas12a and MSREs provide a high level of versatility to the Cas12a-MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.


title: Massively parallel pooled screening reveals genomic determinants of nanoparticle-cell interactions

abstract: To accelerate the translation of cancer nanomedicine, we hypothesize that integrated genomic screens will improve understanding of the cellular processes governing nanoparticle trafficking. We developed a massively parallel high-throughput screening method leveraging barcoded, pooled cancer cell lines annotated with multi-omic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. This approach identified both the materials properties and cell-intrinsic features mediating nanoparticle-cell association. Coupling the data with machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers, including gene expression of SLC46A3. We engineered cell lines to validate SLC46A3 as a biomarker whose expression inversely predicts liposomal nanoparticle uptake both in vitro and in vivo. We further demonstrated the predictive capabilities extend beyond liposomal nanoparticles, regulating both uptake and transfection efficacy of solid lipid nanoparticles. Our work establishes the power of massively parallel pooled cell screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations for specific patient populations.

category: bioengineering


title: ECNano: A Cost-Effective Workflow for Target Enrichment Sequencing and Accurate Variant Calling on 4,800 Clinically Significant Genes Using a Single MinION Flowcell

abstract:
BackgroundThe application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample.

MethodWe introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4,800 clinically important genes and regions using a single MinION flowcell. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples.

ResultsECNano achieved deep on-target depth of coverage (DoC) at average >100x and >98% uniformity using one MinION flowcell. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30x DoC. ECNano obtained an average read length of 1,000 bp. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having [≥] 30x DoC. Clair-ensemble achieved >99% recall and accuracy for SNV calling. The whole workflow from wet-lab protocol to variant detection was completed within three days.

ConclusionWe presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4,800 clinically significant genes and regions using a single MinION flowcell. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction.


title: MSABrowser: dynamic and fast visualization of sequence alignments, variations, and annotations

abstract:
Sequence alignment is an excellent way to visualize the similarities and differences between DNA, RNA, or protein sequences, yet it is currently difficult to jointly view sequence alignment data with genetic variations, modifications such as post-translational modifications, and annotations (i.e. protein domains). Here, we develop the MSABrowser tool that makes it easy to co-visualize genetic variations, modifications, and annotations on the respective positions of amino acids or nucleotides in pairwise or multiple sequence alignments. MSABrowser is developed entirely in JavaScript and works on any modern web browser at any platform, including Linux, Mac OS X, and Windows systems without any installation. MSABrowser is also freely available for the benefit of the scientific community.

Availability and implementationMSABrowser is released as open-source and web-based software under GNU General Public License, version 3.0 (GPLv3). The visualizer, documentation, all source codes, and examples are available at http://thekaplanlab.github.io/ and GitHub repository https://github.com/thekaplanlab/msabrowser.

Supplementary informationSupplementary data are available online.


title: Structural basis for how sMAC is packaged for clearance

abstract: Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane {beta}-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.

category: biophysics


title: Multi-Scale Coarse Grained Model for the Stepping of Molecular Motors with application to Kinesin.

abstract: Conventional kinesin, a motor protein that transports cargo within cells, walks by taking multiple steps towards the plus end of the microtubule (MT). While significant progress has been made in understanding the details of the walking mechanism of kinesin there are many unresolved issues. From a computational perspective, a central challenge is the large size of the system, which limits the scope of time scales accessible in standard computer simulations. Here, we create a general multi-scale coarse-grained model for motors that enables us to simulate the stepping process of motors on polar tracks (actin and MT) with focus on kinesin. Our approach greatly shortens the computation times without a significant loss in detail, thus allowing us to better describe the molecular basis of the stepping kinetics. The small number of parameters, which are determined by fitting to experimental data, allows us to develop an accurate method that may be adopted to simulate stepping in other molecular motors. The model enables us to simulate a large number of steps, which was not possible previously. We show in agreement with experiments that due to the docking of the neck linker (NL) of kinesin, sometimes deemed as the power stroke, the space explored diffusively by the tethered head is severely restricted allowing the step to be in a tens of microseconds. We predict that increasing the interaction strength between the NL and the motor head, achievable by mutations in the NL, decreases the stepping time but reaches a saturation value. Furthermore, the full 3-dimensional dynamics of the cargo are fully resolved in our model, contributing to the predictive power and allowing us to study the important aspects of cargo-motor interactions.


title: Defined diets for freshwater planarians

abstract: Planarian flatworms are popular invertebrate models for basic research on stem cell biology and regeneration. These animals are commonly maintained on a diet of homogenized calf liver or boiled egg yolk in the laboratory, introducing a source of uncontrolled experimental variability. Here, we report the development of defined diets, prepared entirely from standardized, commercially sourced ingredients, for the freshwater species Schmidtea mediterranea, Dugesia japonica, and Girardia dorotocephala. These food sources provide an opportunity to test the effects of specific nutritional variables on biological phenomena of interest. Defined diet consumption was not sufficient for growth and only partially induced the increase in stem cell division that normally accompanies feeding, suggesting these responses are not solely determined by caloric intake. While our defined diet formulations do not support long-term planarian maintenance, they do enable delivery of double-stranded RNA for gene knockdown in a manner that provides unique advantages in some experimental contexts. We also present a new approach for preserving tissue integrity during hydrogen peroxide bleaching of liver-fed animals. These tools will empower research on the connections between diet, metabolism, and stem cell biology in the experimentally tractable planarian system.

category: physiology


title: cellxgene: a performant, scalable exploration platform for high dimensional sparse matrices

abstract:
Quickly and flexibly exploring high-dimensional datasets, such as scRNAseq data, is underserved but critical for hypothesis generation, dataset annotation, publication, sharing, and community reuse. cellxgene is a highly generalizable, web-based interface for exploring high dimensional datasets along categorical, continuous and spatial dimensions, as well as feature annotation. cellxgene is differentiated by its ability to performantly handle millions of observations, and bridges a critical gap by enabling computational and experimental biologists to iteratively ask questions of private and public datasets. In doing so, cellxgene increases the utility and reusability of datasets across the single-cell ecosystem.

The codebase can be accessed at https://github.com/chanzuckerberg/cellxgene. For questions and inquiries, please contact cellxgene@chanzuckerberg.com.


title: Novel antiviral interferon sensitive genes unveiled by correlation-driven gene selection and integrated systems biology approaches.

abstract: Interferons (IFNs) are key cytokines involved in alerting the immune system to viral infection. After IFN stimulation, cellular transcriptional profile critically changes, leading to the expression of several IFN stimulated genes (ISGs) that exert a wide variety of antiviral activities. Despite many ISGs have been already identified, a comprehensive network of coding and non-coding genes with a central role in IFN-response still needs to be elucidated. We performed a global RNA-Seq transcriptome profile of the HCV permissive human hepatoma cell line Huh7.5 and its parental cell line Huh7, upon IFN treatment, to define a network of genes whose coordinated modulation plays a central role in IFN-response. Our study adds molecular actors, coding and non-coding genes, to the complex molecular network underlying IFN-response and shows how systems biology approaches, such as correlation networks, networks topology and gene ontology analyses can be leveraged to this aim.


title: Dynamic task-belief is an integral part of decision-making

abstract: Natural decisions involve two seemingly separable processes: inferring the relevant task (task-belief) and performing the believed-relevant task. The assumed separability has led to the traditional practice of studying task-switching and perceptual decision-making individually. Here, we used a novel paradigm to manipulate and measure macaque monkeys task-belief, and demonstrated inextricable neuronal links between flexible task-belief and perceptual decision-making. We showed that in animals, but not artificial networks that performed as well or better than the animals, stronger task-belief is associated with better perception. Correspondingly, recordings from neuronal populations in cortical areas 7a and V1 revealed that stronger task-belief is associated with better discriminability of the believed-relevant but not the believed-irrelevant feature. Perception also impacts belief updating: noise fluctuations in V1 help explain how task-belief is updated. Our results demonstrate that complex tasks and multi-area recordings can reveal fundamentally new principles of how biology affects behavior in health and disease.


title: Perseverative behavior under uncertainty is enhanced by tryptophan depletion but diminished with subclinical obsessive-compulsive symptoms

abstract: The authors have withdrawn their manuscript as the results and interpretation have changed substantially following further analyses. Please do not cite this manuscript in its current form. If you have any questions, please contact the corresponding author.


title: Ripple Band Phase Precession of Place Cell Firing during Replay

abstract: Phase coding offers several theoretical advantages for information transmission compared to an equivalent rate code. Phase coding is shown by place cells in the rodent hippocampal formation, which fire at progressively earlier phases of the movement related 6-12Hz theta rhythm as their spatial receptive fields are traversed. Importantly, however, phase coding is independent of carrier frequency, and so we asked whether it might also be exhibited by place cells during 150-250Hz ripple band activity, when they are thought to replay information to neocortex. We demonstrate that place cells which fire multiple spikes during candidate replay events do so at progressively earlier ripple phases, and that spikes fired across all replay events exhibit a negative relationship between decoded location within the firing field and ripple phase. These results provide insights into the mechanisms underlying phase coding and place cell replay, as well as the neural code propagated to downstream neurons.


title: The neuroprotective effect of meloxicam in a transient ischemia model involves an increase in axonal sprouting but a decrease in new neuron formation after 7 days ofreperfusion

abstract:
The inflammatory response plays an important role in neuroprotection and regeneration after ischemic insult. The use of non-steroidal anti-inflammatory drugs has been a matter of debate as to whether they have beneficial or detrimental effects. In this context, the effects of the anti-inflammatory agent meloxicam have been scarcely documented after stroke, but its ability to inhibit both cyclooxygenase isoforms (1 and 2) could be a promising strategy to modulate post-ischemic inflammation. This study analyzed the effect of the anti-inflammatory agent meloxicam in a transient focal ischemia model in rats, measuring its neuroprotective effect after 48 hours and 7 days of reperfusion and the effects of the treatment on the glial scar and regenerative events such as the generation of new progenitors in the subventricular zone and axonal sprouting at the edge of the damaged area. We show that meloxicams neuroprotective effects remained after 7 days of reperfusion even if its administration was restricted to the two first days after ischemia. Moreover, meloxicam treatment modulated glial scar reactivity, which matched with an increase in axonal sprouting. However, this treatment decreased the formation of neuronal progenitor cells. This study discusses the dual role of anti-inflammatory treatments after stroke and encourages the careful analysis of both the neuroprotective and the regenerative effects in preclinical studies.

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title: Latent functional connectivity underlying multiple brain states

abstract: Functional connectivity (FC) studies have predominantly focused on resting state, where ongoing dynamics are thought to reflect the brains intrinsic network architecture, which is thought to be broadly relevant because it persists across brain states (i.e., is state-general). However, it is unknown whether resting state is the optimal state for measuring intrinsic FC. We propose that latent FC, reflecting shared connectivity patterns across many brain states, better captures state-general intrinsic FC relative to measures derived from resting state alone. We estimated latent FC independently for each connection using leave-one-task-out factor analysis in 7 highly distinct task states (24 conditions) and resting state using fMRI data from the Human Connectome Project. Compared to resting-state connectivity, latent FC improves generalization to held-out brain states, better explaining patterns of connectivity and task-evoked activation. We also found that latent connectivity improved prediction of behavior outside the scanner, indexed by the general intelligence factor (g). Our results suggest that FC patterns shared across many brain states, rather than just resting state, better reflects state-general connectivity. This affirms the notion of "intrinsic" brain network architecture as a set of connectivity properties persistent across brain states, providing an updated conceptual and mathematical framework of intrinsic connectivity as a latent factor.


title: A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling

abstract:
A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported.

In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed.

Author summaryTrypanosoma brucei is the causative agent of African trypanosomiasis (sleeping sickness) in humans and nagana in cattle. Its unusual genomic organisation featuring large polycistronic units requires a general mechanism of transcription initiation, because individual gene promoters are mostly absent. Despite the fact that the histone variant H2A.Z has previously been identified as a key player of transcription regulation, the complex responsible for correct H2A.Z incorporation at transcription start sites (TSS) remains elusive. In other eukaryotes, SWR1, a SNF2 ATPase-associated chromatin remodelling complex, is responsible for correct incorporation of this histone variant. This study identified a SWR1-like complex in T. brucei. Depletion of the SNF2 ATPase resulted in a reduction of H2A.Z incorporation at the TSS and decreased steady-state mRNA levels accompanied by chromatin condensation. In addition to the SWR1-like complex, we also identified a trypanosome-specific HAT2 complex that includes the histone acetyltransferases HAT2, a key player in the H2A.Z incorporation process. This complex has a trypanosome-specific composition that is different from the NuA4/TIP60 complex in Saccharomyces cerevisiae.

category: microbiology


title: Klebsiella pneumoniae BolA contributes to cell morphology, siderophore production, stresses challenge, cell adhesion and virulence

abstract:
Klebsiella pneumoniae infection is one of the important reasons for the increased of morbidity and mortality. The main virulence factors of K. pneumoniae include capsule polysaccharide, lipopolysaccharide, fimbriae, outer membrane proteins and siderophores. BolA homologues form a broadly conserved family of proteins in prokaryotes and eukaryotes. In Escherichia coli, bolA expression is quickly induced in response to different stresses or stationary phase that rapidly adapt to changing environments. In this report, we confirmed that bolA mutant strain exhibited increased sensitivity to bile and oxidative stresses. In addition, gene deletion showed that bolA has an important role for the adherence of K. pneumoniae to host cell and establishment in mice, including liver, spleen, kidney and lung tissues, and induce the formation of liver abscess in mice. Our results also demonstrated that K. pneumoniae bolA increases the production of siderophore and virulence in Galleria mellonella larvae. Collectively, our results demonstrated that K. pneumoniae BolA is a new virulence factor which contributes to survival in different stresses and overcome host defense. These findings are helpful for the research of new treatment strategies for K. pneumoniae infection.

IMPORTANCEKlebsiella pneumoniae is an important conditional pathogen causing nosocomial infections and community-acquired infections. It can resistant to multiple antibiotics, causing refractory infections and public health threat. Therefore, new treatments are required to fight the pathogen, and a better understanding of its virulence factors are needed to develop new drugs. Here, we unraveled the role of BolA in survival under different stresses and overcome host defense. Our results suggested that bolA actively contributes to cell morphology, stresses challenge, cell adhesion and siderophore production that are tightly related to bacterial virulence. Therefore, bolA mutant strain reduces the virulence of K. pneumoniae in G. mellonella larvae and its colonization ability in mice. These results reported bolA is a key virulence factor in K. pneumoniae, and they are helpful for research of new therapies to treat this increasingly problematic pathogen.


title: An ecological niche model to predict the geographic distribution of Haemagogus janthinomys, Dyar, 1921 the yellow fever and Mayaro virus vector, in South America

abstract:
Yellow fever virus (YFV) has a long history of impacting human health in South America. Mayaro virus (MAYV) is an emerging arbovirus of public health concern in the Neotropics and its full impact is yet unknown. Both YFV and MAYV are primarily maintained via a sylvatic transmission cycle but can be opportunistically transmitted to humans by the bites of infected forest dwelling Haemagogus janthinomys Dyar, 1921. To better understand the potential risk of YFV and MAYV transmission to humans, a more detailed understanding of this vector species distribution is critical. This study compiled a comprehensive database of 170 unique Hg. janthinomys collection sites retrieved from the published literature, digitized museum specimens and publicly accessible mosquito surveillance data. Covariate analysis was performed to optimize a selection of environmental (topographic and bioclimatic) variables associated with predicting habitat suitability, and species distributions modelled across South America using a maximum entropy (MaxEnt) approach. Our results indicate that suitable habitat for Hg. janthinomys can be found across forested regions of South America including the Atlantic forests and interior Amazon.

Author SummaryMayaro virus is a neglected tropical disease and there is insufficient evidence to define its geographic range. The mosquito Haemagogus janthinomys is a primary vector of Mayaro and its distribution is largely unknown at a sub-country scale. Building compendiums of collection data and creating ecological niche models provides a more precise estimation vector species potential habitat. Our dataset stands as one of the most expansive existing for collection data of this species combining data published in literature, publicly available data repositories and digitized museum specimen records. Comparing results of niche models with near real time environmental data can give even better predictions of areas where Mayaro virus exposure could occur. The methods and results of this study can be replicated for any disease/vector of interest so long as there is data discoverable through the scientific literature, public repositories, or other civilian and governmental agencies willing to share.

category: ecology


title: Interspecies recombination has driven the macroevolution of cassava mosaic begomoviruses

abstract:
Begomoviruses (family Geminiviridae, genus Begomovirus) significantly hamper crop production and threaten food security around the world. The frequent emergence of new begomovirus genotypes is facilitated by high mutation frequencies and the propensity to recombine and reassort. Homologous recombination has been especially implicated in the emergence of novel cassava mosaic begomovirus (CMB) genotypes, which cause cassava mosaic disease (CMD). Cassava (Manihot esculenta) is a staple food crop throughout Africa, and an important industrial crop in Asia, two continents where production is severely constrained by CMD. The CMD species complex is comprised of 11 bipartite begomovirus species with ample distribution throughout Africa and the Indian subcontinent. While recombination is regarded as a frequent occurrence for CMBs, a revised, systematic assessment of recombination and its impact on CMB phylogeny is currently lacking. We assembled datasets of all publicly available, full-length DNA-A (n=880) and DNA-B (n=369) nucleotide sequences from the 11 recognized CMB species. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the CMB sequences. Six out of the eleven species have descended from unique interspecies recombination events. Estimates of recombination and mutation rates revealed that all species experience mutation more frequently than recombination, but measures of population divergence indicate that recombination is largely responsible for the genetic differences between species. Our results support that recombination has significantly impacted the CMB phylogeny and is driving speciation in the CMD species complex.

IMPORTANCECassava mosaic disease (CMD) is a significant threat to cassava production throughout Africa and Asia. CMD is caused by a complex comprised of 11 recognized virus species exhibiting accelerated rates of evolution, driven by high frequencies of mutation and genetic exchange. Here, we present a systematic analysis of the contribution of genetic exchange to cassava mosaic virus diversity. Most of these species emerged as a result of genetic exchange. This is the first study to report the significant impact of genetic exchange on speciation in a group of viruses.


title: Species-specific gene duplication in Arabidopsis thaliana evolved novel phenotypic effects on morphological traits under strong positive selection

abstract: Gene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, the ways in which new duplicate genes contribute to phenotypic evolution remain largely unknown, especially in plants, owing to a lack of experimental and phenotypic data. In this study, we identified the new gene Exov, derived from a partial gene region duplication of its parental gene Exov-L, which is a member of an exonuclease family, into a different chromosome in Arabidopsis thaliana. We experimentally investigated the phenotypic effects of Exov and Exov-L in an attempt to understand how the new gene diverged from the parental copy and contributes to phenotypic evolution. Evolutionary analysis demonstrated that Exov is a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNAseq analyses reveal that the new gene, despite its young age, has acquired a large number of novel direct and indirect interactions in which the parental gene does not engage. This is consistent with a high, selection-driven substitution rate in the protein sequence encoded by Exov in contrast to the slowly evolving Exov-L, suggesting an important role for Exov in phenotypic evolution. We analyzed phenotypic effects of exov and exov-l single T-DNA-insertion mutants;double exov, exov-l T-DNA insertion mutants; and CRISPR/Cas9-mediated exovcrp and exov-lcrp knockouts on seven morphological traits in both the new and parental genes. We detected significant segregation of morphological changes for all seven traits when assessed in terms of single mutants, as well as morphological changes for seven traits associated with segregation of double exov, exov-l mutants. Substantial divergence of phenotypic effects between new and parental genes was revealed by principal component analyses, suggesting neofunctionalization in the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological and developmental evolution in Arabidopsis thaliana.