title: Sufu regulation of Hedgehog signaling in P19 cells is required for proper glial cell differentiation

abstract: Hedgehog signaling is essential for vertebrate development, however, less is known about the negative regulators that influence this pathway during the differentiation of cell fates. Using the mouse P19 embryonal carcinoma cell model, Suppressor of Fused (SUFU), a negative regulator of the Hedgehog pathway, was investigated during retinoic acid-induced neural differentiation. We found Hedgehog signaling was activated in the early phase of neural differentiation but was inactive during terminal differentiation of neurons and astrocytes. This early activation was required for neural differentiation however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of GLI, remained relatively unchanged during the differentiation process, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hedgehog target genes. Interestingly, these SUFU-deficient cells were unable to differentiate in the absence of retinoic acid, but when differentiated using retinoic acid, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout retinoic acid-induced differentiation and this phenotype was accompanied by the loss of GLI3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, and its normal regulation of GLI3 to maintain Hh signaling in an inactive state.


title: Sufu regulation of Hedgehog signaling in P19 cells is required for proper astrocyte differentiation

category: developmental biology


title: Multi-organ analysis of low-level somatic mosaicism reveals stage- and tissue-specific mutational features in human development

abstract: Most somatic mutations arising during normal development present as low-level in single or multiple tissues depending on the developmental stage and affected organs1-4. However, it remains unclear how the human developmental stages or mutation-carrying organs affect somatic mutations features. Here, we performed a systemic and comprehensive analysis of low-level somatic mutations using deep whole-exome sequencing (WES; average read depth: [~]500x) of 498 multiple organ tissues with matched controls from 190 individuals. We found that early-stage mutations shared between multiple organs are lower in number but showed higher allele frequencies than late-stage mutations [0.54 vs. 5.83 variants per individual: 6.17% vs. 1.5% variant allele frequency (VAF)] along with less nonsynonymous mutations and lower functional impacts. Additionally, early- and late-stage mutations had unique mutational signatures distinct from tumor-originate mutations. Compared with early-stage mutations presenting a clock-like signature across all studied organs or tissues, late-stage mutations show organ, tissue, and cell-type specificity in mutation count, VAFs, and mutational signatures. In particular, analysis of brain somatic mutations shows bimodal occurrence and temporal-lobe-specific mutational signatures. These findings provide new insight into the features of somatic mosaicisms dependent on developmental stages and brain regions.

category: genomics


title: Neuronal control of suppression, initiation and completion of egg deposition in Drosophila melanogaster

abstract: Egg-laying in Drosophila is the product of post-mating physiological and behavioural changes that culminate in a stereotyped sequence of actions. While egg-laying behaviour has been mostly used as a system to understand the neuronal basis of decision making in the context of site selection, it harbours a great potential as a paradigm to uncover how, once a site is selected, the appropriate motor circuits are organized and activated to deposit an egg. To study this programme, we first describe the different stages of the egg-laying programme and the specific actions associated with each stage. Using a combination of neuronal activation and silencing experiments we characterize the role of three distinct neuronal populations in the abdominal ganglion with different contributions to the egg deposition motor elements. Specifically, we identify a subset of glutamatergic neurons and a subset of cholinergic neurons that promote the initiation and completion of egg expulsion respectively, while a subset of GABAergic neurons suppresses egg-laying. This study provides insight into the organization of neuronal circuits underlying complex motor behaviour.

category: neuroscience


title: TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

abstract: The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimers disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum (ER) stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.


title: Superovulation does not alter calcium oscillations following fertilization

abstract: Superovulation is a common approach to maximize the number of eggs available for either clinical assisted reproductive technologies or experimental animal studies. This procedure provides supraphysiological amounts of gonadotropins to promote continued growth and maturation of ovarian follicles that otherwise would undergo atresia. There is evidence in mice, cows, sheep and humans that superovulation has a detrimental impact on the quality of the resulting ovulated eggs or embryos. Here we tested the hypothesis that eggs derived from superovulation have a reduced capacity to support calcium oscillations following fertilization, which is a critical factor in the success of embryo development. Eggs were obtained from mice that were either naturally cycling or underwent a standard superovulation protocol. Naturally cycling mice were mated to vasectomized males and vaginal plugs were checked to assure ovulation had occurred. The superovulated mice were also mated to vasectomized males for consistency of treatment across groups. The eggs were fertilized in vitro while undergoing monitoring of calcium oscillatory patterns. There were no differences in any measures of calcium oscillatory behavior, including length of the first oscillation, area under the curve of calcium signal, or frequency or persistence of oscillations. These findings indicate that superovulation does not disrupt calcium signaling at fertilization, supporting the use of this method for both clinical and experimental purposes.


title: Profiling Transcription Initiation in Peripheral Leukocytes Reveals Severity-Associated Cis-Regulatory Elements in Critical COVID-19

abstract: The contribution of transcription factors (TFs) and gene regulatory programs in the immune response to COVID-19 and their relationship to disease outcome is not fully understood. Analysis of genome-wide changes in transcription at both promoter-proximal and distal cis-regulatory DNA elements, collectively termed the active cistrome, offers an unbiased assessment of TF activity identifying key pathways regulated in homeostasis or disease. Here, we profiled the active cistrome from peripheral leukocytes of critically ill COVID-19 patients to identify major regulatory programs and their dynamics during SARS-CoV-2 associated acute respiratory distress syndrome (ARDS). We identified TF motifs that track the severity of COVID- 19 lung injury, disease resolution, and outcome. We used unbiased clustering to reveal distinct cistrome subsets delineating the regulation of pathways, cell types, and the combinatorial activity of TFs. We found critical roles for regulatory networks driven by stimulus and lineage determining TFs, showing that STAT and E2F/MYB regulatory programs targeting myeloid cells are activated in patients with poor disease outcomes and associated with single nucleotide genetic variants implicated in COVID-19 susceptibility. Integration with single-cell RNA-seq found that STAT and E2F/MYB activation converged in specific neutrophils subset found in patients with severe disease. Collectively we demonstrate that cistrome analysis facilitates insight into disease mechanisms and provides an unbiased approach to evaluate global changes in transcription factor activity and stratify patient disease severity.


title: SEA: Simple Enrichment Analysis of motifs

abstract: Motif enrichment algorithms can identify known sequence motifs that are present to a statistically significant degree in DNA, RNA and protein sequences. Databases of such known motifs exist for DNA- and RNA-binding proteins, as well as for many functional protein motifs. The SEA ("Simple Enrichment Analysis") algorithm presented here uses a simple, consistent approach for detecting the enrichment of motifs in DNA, RNA or protein sequences, as well as in sequences using user-defined alphabets. SEA can identify known motifs that are enriched in a single set of input sequences, and can also perform differential motif enrichment analysis when presented with an additional set of control sequences. Using in vivo DNA (ChIP-seq) data as input to SEA, and validating motifs with reference motifs derived from in vitro data, we show that SEA is is faster than three widely-used motif enrichment algorithms (AME, CentriMo and Pscan), while delivering comparable accuracy. We also show that, in contrast to other motif enrichment algorithms, SEA reports accurate estimates of statistical significance. SEA is easy to use via its web server at https://meme-suite.org, and is fully integrated with the widely-used MEME Suite of sequence analysis tools, which can be freely downloaded at the same web site for non-commercial use.

category: bioinformatics


title: proovframe: frameshift-correction for long-read (meta)genomics

abstract: Long-read sequencing technologies hold big promises for the genomic analysis of complex samples such as microbial communities. Yet, despite improving accuracy, basic gene prediction on long-read data is still often impaired by frameshifts resulting from small indels. Consensus polishing using either complementary short reads or to a lesser extent the long reads themselves can mitigate this effect but requires universally high sequencing depth, which is difficult to achieve in complex samples where the majority of community members are rare. Here we present proovframe, a software implementing an alternative approach to overcome frameshift errors in long-read assemblies and raw long reads. We utilize protein-to-nucleotide alignments against reference databases to pinpoint indels in contigs or reads and correct them by deleting or inserting 1-2 bases, thereby conservatively restoring reading-frame fidelity in aligned regions. Using simulated and real-world benchmark data we show that proovframe performs comparably to short-read-based polishing on assembled data, works well with remote protein homologs, and can even be applied to raw reads directly. Together, our results demonstrate that protein-guided frameshift correction significantly improves the analyzability of long-read data both in combination with and as an alternative to common polishing strategies. Proovframe is available from https://github.com/thackl/proovframe.


title: Counterbalance of Foxp3 and IDO expression at different tumor stages in aggressive breast cancer subtypes

abstract:
Foxp3 and IDO1 are known immunomodulatory molecules involved in tumor escape and could be related to tumor infiltrating lymphocytes (TILs) in the tumor microenvironment. In this study, tumoral Foxp3 and IDO1 expression in breast cancer were evaluated in relation to lymphocyte biomarkers such as CD8 and CD45R0, regulatory T cells, as well as intratumoral and stromal TILs (iTILs and sTILs, respectively). Clinical and histopathological features were also included in the analysis. Foxp3 and IDO1 were found in tumor cells showing mainly cytoplasmic patterns in 60% and 62% tumor samples, respectively. TILs were found in 76% of samples; iTILs were detected in 92% of those samples and sTILs in 55%. Foxp3+ TILs were detected only in 12% of TILs+ samples associated with tumoral Foxp3 expression. Tumoral Foxp3 was mainly expressed at lower tumor stages while IDO1 expression was associated with advanced tumor stages; both correlated with CD8+ TILs which were observed in 77% of TILs+ samples. CD45R0+ were observed in 81% of TILs+ samples and correlated with higher tumor stages and poorly differentiated tumors. In ER negative tumors, an inverse correlation between Foxp3 and IDO1 tumoral expression was found in relation to tumor stage. TNBC subtype showed a positive correlation with the presence of iTILs. In silico analysis showed that Foxp3 and coexpressed genes in breast cancer were associated with immune response genes. Foxp3 was found predominantly in Basal and Her2-enriched subtypes in relation to Luminal A subtype, by RNA seq and RNA microarray database analysis. In conclusion, the expression of Foxp3 and IDO1 in tumors at different stages suggests a potential compensatory mechanism to evade the strong CTL response observed. This is relevant since the cumulative data indicates that Foxp3 as well as IDO1 could be potential targets of immunotherapy in patients with tumors at different stages and for the most aggressive breast cancer subtypes such as TNBC and Her2-enriched.

HIGHLIGHTSO_LIFoxp3 and IDO1 were found overexpressed in the cytoplasm of tumor cells, observed in BC samples at early or advanced tumor stages, respectively, both associated with abundant cytotoxic CD8+ lymphocytes infiltrates.
C_LIO_LITumor immunosuppression by Foxp3 and IDO1 tumoral expression could counteract the attempt of the hosts immune system to eradicate the tumor by CD8+ and CD45R0+ TILs cytotoxic activity.
C_LIO_LIIt is well-known the tolerogenic role of Foxp3 in Treg and less understood the role of Foxp3 in tumors. However, there is increasing evidence that Foxp3+ tumor cells appear to promote an immunosuppressive microenvironment similar to Treg. Whether this is a consequence of the effect of other immunosuppressive factors secreted by the tumor cells which can be induced by Foxp3 expression, such as TGF{beta}, remains to be determined.
C_LI

category: cancer biology


title: Experimental safety testing shows that the NSAID tolfenamic acid is not toxic to Gyps vultures in India at concentrations likely to be encountered in cattle carcasses

abstract: Population declines of Gyps vultures across the Indian subcontinent were caused by unintentional poisoning by the non-steroidal anti-inflammatory drug (NSAID) diclofenac. Subsequently, a number of other NSAIDs have been identified as toxic to vultures, while one, meloxicam, is safe at concentrations likely to be encountered by vultures in the wild. Other vulture-safe drugs need to be identified to reduce the use of those toxic to vultures. We report on safety-testing experiments on the NSAID tolfenamic acid on captive vultures of three Gyps species, all of which are susceptible to diclofenac poisoning. Firstly, we estimated the maximum level of exposure (MLE) of wild vultures and gave this dose to 38 Near Threatened G. himalayensis by oral gavage, with 15 control birds dosed with benzyl alcohol (the carrier solution for tolfenamic acid). Two birds given tolfenamic acid died with elevated uric acid levels and severe visceral gout, while the remainder showed no adverse clinical or biochemical signs. Secondly, four G. himalayensis were fed tissues from water buffaloes which had been treated with double the recommended veterinary dose of tolfenamic acid prior to death and compared to two birds fed uncontaminated tissue; none suffered any clinical effects. Finally, two captive Critically Endangered vultures, one G. bengalensis and one G. Indicus, were given the MLE dose by gavage and compared to two control birds; again, none suffered any clinical effects. The death of two G. himalayensis may have been an anomaly due to i) the high dose level used and ii) the high ambient temperatures at the time of the experiment. Tolfenamic acid is likely to be safe to Gyps vultures at concentrations encountered by wild birds and could therefore be promoted as a safe alternative to toxic NSAIDs. It is manufactured in the region, and is increasingly being used to treat livestock.

category: pharmacology and toxicology


title: A compilation of fecal microbiome shotgun metagenomics from hospitalized patients undergoing hematopoietic cell transplantation

abstract: Hospitalized patients receiving hematopoietic cell transplants provide a unique opportunity to study how the human gut microbiome changes in response to perturbations, and how the resulting changes in the microbiome feedback on its living host. We previously compiled a large-scale longitudinal dataset of stool microbiome compositions from these patients and associated metadata1. In that dataset the microbiome analysis was limited to the taxonomic composition of the bacterial population obtained from 16S rRNA gene sequencing. Here, we augment those data with shotgun metagenomic sequences from a nested subset of 395 stool samples. We provide accession numbers that link each sample to the paired-end sequencing files deposited in a public repository, which can be directly accessed by the online services of PATRIC2 to be analyzed without the users having to download or transfer the files. We provide examples that show how shotgun sequencing enriches microbiome analyses beyond the taxonomic composition such as the analysis of gene functions including virulence factors and antibiotic resistances, and the assembly of genomes from metagenomic data.

category: systems biology


title: CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states

abstract: In response to central nervous system injury or disease, astrocytes become reactive, adopting context-dependent states with altered functions. Certain inflammatory insults induce reactive astrocyte states that lose homeostatic functions and gain harmful outputs, and likely contribute to neuroinflammatory and neurodegenerative diseases. However, the cellular pathways controlling these states are not fully understood. Here, we combined single-cell transcriptomics with CRISPRi screening in human iPSC-derived astrocytes to systematically interrogate inflammatory reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-{kappa}B activation drove two distinct inflammatory reactive states promoted by and inhibited by STAT3, respectively. Furthermore, these states corresponded with those observed in other experimental contexts, including in vivo, and their markers were upregulated in the human brain in Alzheimers disease and hypoxic ischemic encephalopathy. These results and the platform we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.


title: Beyond body maps: information content of specific body parts is distributed across the somatosensory Homunculus

abstract: The somatosensory homunculus in primary somatosensory cortex (S1) is topographically organised, with relatively high selectivity to each body part in its primary area. This dominant feature may eclipse other organising principles in S1. Recent multivariate methodologies allow us to identify representational features beyond selectivity, e.g., information content, providing new opportunities to characterise the homunculus. Using Representational Similarity Analysis, we asked whether body part information content can be identified in S1 beyond the primary area of a given body part. Representational dissimilarities in fMRI activity patterns were compared between different body parts (face, hand and feet) and subparts (e.g., fingers), and between different actions performed with the same body part. Throughout the S1 homunculus, we identified significant dissimilarities between non-primary body parts (e.g., between the hand and the lips in the foot area). We also observed significant dissimilarities between body subparts in distant non-primary areas (e.g., different face parts in the foot area). Finally, we could significantly dissociate between two movements performed by one body part (e.g., the hand) well beyond its primary area (e.g., in the foot and face areas), even when focusing the analysis along the most topographically organised sub-region of S1, Brodmann area 3b. Altogether, our results demonstrate that body part and action-related information content is more distributed across S1 homunculus than previously considered. While this finding does not revoke the general topographic organising principle of S1, it reveals yet unexplored underlying information contents that could be harnessed for rehabilitation, as well as novel brain-machine interfaces.


title: LRRK2 causes centrosomal deficits via phosphorylated Rab10 and RILPL1 at centriolar subdistal appendages

abstract: The Parkinsons disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that various LRRK2 risk variants and all currently described regulators of the LRRK2 signaling pathway converge upon causing centrosomal cohesion deficits. The cohesion deficits do not require the presence of RILPL2 or of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendages of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab protein to cause the centrosomal defects. These data reveal a common molecular pathway by which alterations in the LRRK2 kinase activity impact upon centrosome-related events.


title: Structure-based design of antisense oligonucleotides that inhibit SARS-CoV-2 replication

abstract: Antisense oligonucleotides (ASOs) are an emerging class of drugs that target RNAs. Current ASO designs strictly follow the rule of Watson-Crick base pairing along target sequences. However, RNAs often fold into structures that interfere with ASO hybridization. Here we developed a structure-based ASO design method and applied it to target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our method makes sure that ASO binding is compatible with target structures in three-dimensional (3D) space by employing structural design templates. These 3D-ASOs recognize the shapes and hydrogen bonding patterns of targets via tertiary interactions, achieving enhanced affinity and specificity. We designed 3D-ASOs that bind to the frameshift stimulation element and transcription regulatory sequence of SARS-CoV-2 and identified lead ASOs that strongly inhibit viral replication in human cells. We further optimized the lead sequences and characterized structure-activity relationship. The 3D-ASO technology helps fight coronavirus disease-2019 and is broadly applicable to ASO drug development.

category: molecular biology


title: Cell Sex and Sex Hormones Modulate Kidney Glucose and Glutamine Metabolism in Health and Diabetes

abstract: Male sex is a risk factor for progression of diabetic kidney disease, but the reasons for this predilection are unclear. Here, we demonstrate that cell sex and sex hormones alter the metabolic phenotype of human proximal tubular epithelial cells (PTECs). Male PTECs displayed increased glycolysis, mitochondrial respiration, oxidative stress, apoptosis, and high glucose-induced injury, compared to female PTECs. This phenotype was enhanced by dihydrotestosterone (DHT) and linked to increased mitochondrial utilization of glucose and glutamine. Studies in vivo pointed towards increased glutamine anaplerosis in diabetic male kidneys. Male sex was linked to increased levels of glutamate, TCA cycle, and glutathione cycle metabolites, in PTECs and in the blood metabolome of healthy youth and youth with type 2 diabetes. Conversely, female PTECs displayed increased levels of pyruvate, glutamyl-cysteine, cysteinylglycine, and a higher GSH/GSSG ratio than male PTECs, indicative of enhanced redox homeostasis. Finally, we identified transcriptional mechanisms that control kidney metabolism in a sex-specific fashion. While X-linked demethylase KDM6A facilitated metabolic homeostasis in female PTECs, transcription factor HNF4A mediated the deleterious effects of DHT in male PTECs. This work uncovers the role of sex in glucose/glutamine metabolism, that may explain the roots of sex dimorphism in the healthy and diabetic kidney.

category: cell biology


title: Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration

abstract: The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in the case of zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with 5 of 6 elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in hearts of larval animals. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration.


title: Comparison of Single-Trait and Multi-Trait Genome-Wide Association Models and Inclusion of Correlated Traits in the Dissection of the Genetic Architecture of a Complex Trait in a Breeding Program

abstract: Traits with an unknown genetic architecture make it difficult to create a useful bi-parental mapping population to characterize the genetic basis of the trait due to a combination of complex and pleiotropic effects. Seedling emergence of wheat (Triticum aestivum L.) from deep planting is a vital factor affecting stand establishment and grain yield, has a poorly understood genetic architecture, and is historically correlated with coleoptile length. The creation of bi-parental mapping populations can be overcome by using genome-wide association studies (GWAS). This study aimed to dissect the genetic architecture of seedling emergence while accounting for correlated traits using one multi-trait GWAS model (MT-GWAS) and three single-trait GWAS models (ST-GWAS) with the inclusion of covariates for correlated traits. The ST-GWAS models included one single locus model (MLM), and two multiple loci models (FarmCPU and BLINK). We conducted the GWAS using two populations, the first consisting of 473 varieties from a diverse association mapping panel (DP) phenotyped from 2015-2019, and the other population used as a validation population consisting of 279 breeding lines (BL) phenotyped in 2015 in Lind, WA, with 40,368 markers. We also compared the inclusion of coleoptile length and markers associated with reduced height as covariates in our ST-GWAS models for the DP. ST-GWAS found 107 significant markers across 19 chromosomes, while MT-GWAS found 82 significant markers across 14 chromosomes. MT-GWAS models were able to identify large-effect markers on chromosome 5A. FarmCPU and BLINK models were able to identify many small effect markers, and the inclusion of covariates helped to identify the large effect markers on chromosome 5A. Therefore, by using multi-locus models combined with pleiotropic covariates, breeding programs can uncover the complex nature of traits to help identify candidate genes and the underlying architecture of a trait, such as seedling emergence of deep-sown winter wheat.

category: genetics


title: Rates of convergence in the two-island andisolation-with-migration models

abstract: A number of powerful demographic inference methods have been developed in recent years, with the goal of fitting rich evolutionary models to genetic data obtained from many populations. In this paper we investigate the statistical performance of these methods in the specific case where there is continuous migration between populations. Compared with earlier work, migration significantly complicates the theoretical analysis and demands new techniques. We employ the theories of phase-type distributions and concentration of measure in order to study the two-island and isolation-with-migration models, resulting in both upper and lower bounds. For the upper bounds, we consider inferring rates of coalescent and migration on the basis of directly observing pairwise coalescent times, and, more realistically, when (conditionally) Poisson-distributed mutations dropped on latent trees are observed. We complement these upper bounds with information-theoretic lower bounds which establish a limit, in terms of sample size, below which inference is effectively impossible.


title: Converging evidence for differential regulatory control of APOEε4 on African versus European haplotypes

abstract:
INTRODUCTIONThe difference in APOE{varepsilon}4 risk for Alzheimer disease (AD) between different populations is associated with APOE{varepsilon}4 local ancestry (LA). We examined LA SNPs with significant frequency differences between African and European/Japanese APOE{varepsilon}4 haplotypes for areas of differential regulation.

METHODSWe performed two enhancer Massively Parallel Reporter Assay (MPRA) approaches, supplemented with single fragment reporter assays. We utilized Capture C analyses to support interactions with the APOE promoter.

RESULTSThe TOMM40 intron 2 and 3 region showed increased enhancer activity in the European/Japanese versus African LA haplotypes in astrocytes and microglia. This region overlaps with APOE promoter interactions as assessed by Capture C analysis. Single variant analyses pinpoints rs2075650/rs157581, and rs59007384 as functionally different on these haplotypes.

DISCUSSIONBoth differential regulatory function and Capture C data support an intronic region in TOMM40 as contributing to the differential APOE expression between African and European/Japanese LA.


title: Genome assembly, annotation and comparative analysis of the cattail Typha latifolia

abstract: Cattails (Typha species) comprise a genus of emergent wetland plants with a global distribution. Typha latifolia and T. angustifolia are two of the most widespread species, and in areas of sympatry can interbreed to produce the hybrid Typha x glauca. In some regions the relatively high fitness of T. x glauca allows it to outcompete and displace both parent species, while simultaneously reducing plant and invertebrate biodiversity, and modifying nutrient and water cycling. We generated a high-quality whole genome assembly of T. latifolia using PacBio long-read and high coverage Illumina sequences that will facilitate evolutionary and ecological studies in this hybrid zone. Genome size was 287 Mb and consisted of 1,189 scaffolds, with an N50 of 8.706 Mb; 43.84% of the genome were identified as repetitive elements. The assembly has a BUSCO score of 96.03%, and 27,432 genes and 2,700 RNA sequences were putatively identified. Comparative analysis detected over 9,000 shared orthologues with related taxa and phylogenomic analysis supporting Typha latifolia as a divergent lineage within Poales. This high-quality scaffold-level reference genome will provide a useful resource for future population genomic analyses and improve our understanding of Typha hybrid dynamics.


title: Belowground allocation and dynamics of recently fixed plant carbon in a California annual grassland soil

abstract:
Plant roots and the organisms that surround them are a primary source for stabilized organic C, particularly in grassland soils, which have a large capacity to store organic carbon belowground. To quantify the flow and fate of plant fixed carbon (C) in a Northern California annual grassland, we tracked plant carbon from a five-day 13CO2 pulse field labeling for the following two years. Soil and plant samples were collected immediately after the pulse labeling, and again at three days, four weeks, six months, one year, and two years. Soil organic matter was fractionated using a sodium polytungstate density gradient to separate the free-light fraction (FLF), occluded-light fraction (OLF), and heavy fraction (HF). Using isotope ratio mass spectrometry, we measured 13C enrichment and total C content for plant shoots, roots, soil, soil dissolved organic carbon (DOC), and the FLF, OLF, and HF. The HF was further analyzed by solid state 13C NMR spectroscopy.

At the end of the labeling period, the largest amount of 13C was recovered in plant shoots (60%), but a substantial amount (40%) was already found belowground in roots, soil, and soil DOC. Density fractionation of 4-week soil samples (from which living roots were removed) indicated that the highest isotope enrichment was in the mineral-rich heavy fraction, with similar enrichment of the FLF and OLF. At the 6-month sampling, after the dry summer period during which plants senesced and died, the amount of label in the FLF increased such that it was equal to that in the HF. By the 1-year sampling, 13C in the FLF had declined substantially and continued to decline by the 2-year sampling. 13C recovery in the OLF and HF, however, was qualitatively stable between sampling times. By the end of the 2-year experiment, 69% of remaining label was in the HF, 18% in the FLF and 13% in the OLF.

While the total 13C content of the HF did not change significantly from the 4-week to the 2-year sample time, 13C NMR spectroscopic analysis of spring HF samples from 2018, 2019, and 2020 suggests that the relative proportion of aliphatic/alkyl functional groups declined in the newly formed SOC over the 2-year period. Simultaneously, aromatic and carbonyl functional groups increased, and the proportion of carbohydrate groups remained relatively constant. In summary, our results indicate that initial associations between minerals and root-derived organic matter are significant and form rapidly; by 4 weeks, a substantial amount (17%) of the total plant-derived 13C had become associated with the heavy fraction (HF) of soil. While the majority of annual C input cycles rapidly (<2-year timescale), a sizeable proportion ([~]12% of the original inputs) persisted for 2 years.

category: biochemistry


title: Lipid droplet structural remodeling in adipose tissue upon caloric excess

abstract:
Excess calories are stored as triacylglycerols (TAG) and cholesteryl esters (CE) in lipid droplets (LD), and during obesity, LD expansion occurs. X-ray scattering of adipose tissue uncovered that LDs comprise two TAG packing domains: a disordered core and a multilamellar shell. The number of TAG layers increases upon diet-induced obesity and is adipose depot-specific. Further, collagen was highly oriented in brown but randomly dispersed in white fat. We discovered that the bodys surfactant, bile acids (BAs) stimulate remodeling of LD size. Deleting the BA receptor, Farnesoid X receptor (FXR) reduced a hydrophilic BA, {beta} muricholic acid ({beta}-MCA), and enlarged the adipocytes. BA composition is a critical determinant of overall hydrophobicity index and solubilization ability. Accordingly, we found that the obesogenic diet reduced a hydrophobic BA, chenodeoxycholic acid (CDCA). Taken together, these findings implicate that BAs, tissue niches, and diet influence LD structural remodeling.

SummaryLipid droplets (LDs) pack triacylglycerols (TAGs) with altered dimensions and exhibit distinct collagen orientation between the white and brown fat depots and are remodeled by bile acids (BAs) such that deletion of BA-receptor, Farnesoid X receptor (FXR) results in adipocyte hypertrophy.

category: biophysics


title: A deep learning-based iterative digital pathology annotation tool

abstract: Well-annotated exemplars are an important prerequisite for supervised deep learning schemes. Unfortunately, generating these annotations is a cumbersome and laborious process, due to the large amount of time and effort needed. Here we present a deep-learning-based iterative digital pathology annotation tool that is both easy to use by pathologists and easy to integrate into machine vision systems. Our pathology image annotation tool greatly reduces annotation time from hours to a few minutes, while maintaining high fidelity with human-expert manual annotations. Here we demonstrate that our active learning tool can be used for a variety of pathology annotation tasks including masking tumor, stroma, and lymphocyte-rich regions, among others. This annotation automation system was validated on 90 unseen digital pathology images with tumor content from the CAMELYON16 database and it was found that pathologists gold standard masks were re-produced successfully using our tool. That is, an average of 2.7 positive selections (mouse clicks) and 8.0 negative selections (mouse clicks) were sufficient to generate tumor masks similar to pathologists gold standard in CAMELYON16 test WSIs. Furthermore, the developed image annotation tool has been used to build gold standard masks for hundreds of TCGA digital pathology images. This set was used to train a convolutional neural network for identification of tumor epithelium. The developed pan-cancer deep neural network was then tested on TCGA and internal data with comparable performance. The validated pathology image annotation tool described herein has the potential to be of great value in facilitating accurate, rapid pathological analysis of tumor biopsies.

category: pathology


title: Constitutive expression of JASMONATE RESISTANT 1 elevates content of several jasmonates and primes Arabidopsis thaliana to better withstand drought

abstract:
Jasmonates have a well-documented role in balancing the trade-off between plant growth and defense against biotic stresses. However, the role of jasmonate signaling under abiotic stress is less well studied. Here, we investigated the function of JASMONATE RESISTANT 1 (JAR1) in drought stress in Arabidopsis thaliana. JAR1 converts jasmonic acid (JA) to jasmonyl-L-isoleucine (JA-Ile), the major bioactive form of jasmonates. Comparison of a newly generated over-expression line (JAR1-OE) with jar1-11, a T-DNA insertion line in the JAR1 locus, and Col-0 revealed that constitutively increased JA-Ile production results in stunted growth and a delay in flowering. Upon water limitation, JAR1-OE plants retained more water in their leaves, showed reduced wilting and recovered better from drought stress than the wild type. By contrast, jar1-11 mutant plants were hypersensitive to drought. RNA-seq analysis and hormonal profiling of plants under control and drought stress conditions provided insight into the molecular reprogramming caused by the alteration in JA-Ile content. Especially JAR1-OE plants were affected in many adaptive systems related to drought stress, including stomatal density, stomatal aperture or the formation of reactive oxygen species (ROS). Overall, our data suggest that constitutively increased expression of JAR1 can prime Arabidopsis towards improved drought tolerance.

One sentence summaryConstitutive expression of JAR1primes Arabidopsis thaliana to better withstand drought stress

category: plant biology


title: The Chloroflexi supergroup is metabolically diverse and representatives have novel genes for non-photosynthesis based CO2 fixation

abstract: The Chloroflexi superphylum have been investigated primarily from the perspective of reductive dehalogenation of toxic compounds, anaerobic photosynthesis and wastewater treatment, but remain relatively little studied compared to their close relatives within the larger Terrabacteria group, including Cyanobacteria, Actinobacteria, and Firmicutes. Here, we conducted a detailed phylogenetic analysis of the phylum Chloroflexota, the phylogenetically proximal candidate phylum Dormibacteraeota, and a newly defined sibling phylum proposed in the current study, Eulabeiota. These groups routinely root together in phylogenomic analyses, and constitute the Chloroflexi supergroup. Chemoautotrophy is widespread in Chloroflexi. Two Form I Rubisco ancestral subtypes that both lack the small subunit are prevalent in ca. Eulabeiota and Chloroflexota, suggesting that the predominant modern pathway for CO2 fixation evolved in these groups. The single subunit Form I Rubiscos are inferred to have evolved prior to oxygenation of the Earths atmosphere and now predominantly occur in anaerobes. Prevalent in both Chloroflexota and ca. Eulabeiota are capacities related to aerobic oxidation of gases, especially CO and H2. In fact, aerobic and anaerobic CO dehydrogenases are widespread throughout every class-level lineage, whereas traits such as denitrification and reductive dehalogenation are heterogeneously distributed across the supergroup. Interestingly, some Chloroflexota have a novel clade of group 3 NiFe hydrogenases that is phylogenetically distinct from previously reported groups. Overall, the analyses underline the very high level of metabolic diversity in the Chloroflexi supergroup, suggesting the ancestral metabolic platform for this group enabled highly varied adaptation to ecosystems that appeared in the aerobic world.

category: microbiology


title: Transcriptional plasticity drives leukemia immune escape

abstract: Relapse of acute myeloid leukemia (AML) after allogeneic bone marrow transplantation (alloSCT) has been linked to immune evasion due to reduced expression of major histocompatibility complex class II (MHC-II) proteins through unknown mechanisms. We developed CORENODE, a computational algorithm for genome-wide transcription network decomposition, that identified the transcription factors (TFs) IRF8 and MEF2C as positive regulators and MYB and MEIS1 as negative regulators of MHC-II expression in AML cells. We show that reduced MHC-II expression at relapse is transcriptionally driven by combinatorial changes in the levels of these TFs, acting both independently and through the MHC-II coactivator CIITA. Beyond the MHC-II genes, MYB and IRF8 antagonistically regulate a broad genetic program responsible for cytokine signaling and T-cell stimulation that displays reduced expression at relapse. A small number of cells with altered TF levels and silenced MHC-II expression are present at the time of initial leukemia diagnosis, likely contributing to eventual relapse. Our findings reveal an adaptive transcriptional mechanism of AML evolution after allogenic transplantation whereby combinatorial fluctuations of TF levels under immune pressure result in selection of cells with a silenced T-cell stimulation program.


title: Isolation and Genomics of multidrug-resistant Brevundimonas diminuta collected from patients with pertussis-like symptoms

abstract: Brevundinomas diminuta is known as an opportunistic pathogen and is rarely associated with invasive infections in humans causing infection in the different parts of the body. In this study, we identified three B. diminuta isolates from three patients with an initial diagnosis of pertussis. Isolates were confirmed using biochemical tests and 16s rRNA sequencing. All isolates were resistant to three different classes of antimicrobial drugs including ceftazidime and ciprofloxacin. We performed Illumina whole-genome sequencing for two isolates. The results showed an average genome size of 3.25 Mbp with the G+C content 67% with multiple predicted virulence factors and genes leading to antibiotic resistance. We found an open pan-genome with 1502 core genes by analysing 13 available global B. diminuta isolated from different environments such as water, soil, or gentamicin fermentation residue. In the phylogenetic analysis, our isolates were grouped with B. diminuta isolate collected from an oral cavity of a patient in the USA.


title: Cryo-EM structure of the human TACAN channel in a closed state

abstract: TACAN is an ion channel involved in sensing mechanical pain. It has recently been shown to represent a novel and evolutionarily conserved class of mechanosensitive channels. Here, we present the cryoelectron microscopic structure of human TACAN (hTACAN). hTACAN forms a dimer in which each protomer consists of a transmembrane globular domain (TMD) that is formed of six helices and an intracellular domain (ICD) that is formed of two helices. Molecular dynamic simulations suggest that a putative ion conduction pathway is located inside each protomer. Single point mutation of the key residue Met207 significantly increased the surface tension activated currents. Moreover, cholesterols were identified at the flank of each subunit. Our data show the molecular assembly of hTACAN and suggest that the wild type hTACAN is in a closed state, providing a basis for further understanding the activation mechanism of the hTACAN channel.


title: Asymmetric depth-filtration - a versatile and scalable approach for isolation and purification of extracellular vesicles

abstract: A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

category: bioengineering


title: Informative RNA-base embedding for functional RNA structural alignment and clustering by deep representation learning

abstract: Effective embedding is being actively conducted by applying deep learning to biomolecular information. Obtaining better embedding enhances the quality of downstream analysis such as DNA sequence motif detection and protein function prediction. In this study, we adopt a pre-training algorithm for the effective embedding of RNA bases to acquire semantically rich representations, and apply it to two fundamental RNA sequence problems: structural alignment and clustering. By using the pre-learning algorithm to embed the four bases of RNA in a position-dependent manner using a large number of RNA sequences from various RNA families, a context-sensitive embedding representation is obtained. As a result, not only base information but also secondary structure and context information of RNA sequences are embedded for each base. We call this "informative base embedding" and use it to achieve accuracy superior to that of existing state-of-the-art methods in RNA structural alignment and RNA family clustering tasks. Furthermore, by performing RNA sequence alignment combining this informative base embedding with a simple Needleman-Wunsch alignment algorithm, we succeed in calculating a structural alignment in a time complexity O(n2) instead of the O(n6) time complexity of Sankoff-style algorithms.


title: Information Flow in the Fibroblast Growth Factor Receptor Communication Channel

abstract: In this work we analyze the flow of information through the Fibroblast Growth Factor Receptor (FGFR) communication channel when different types of signals are transmitted by the MAPK cascade to the gene regulatory network (GRN) formed by the genes C-Myc, DUSP, and Cdc25A, which control fibroblast proliferation. We used the canonical mathematical model of the MAPK cascade coupled to a stochastic model for the activation of the gene regulatory network, subject to different types of FGF inputs (step, quadratic pulses, Dirac delta, and white noise), in order to analyze the response of the gene regulatory network to each type of signal, and determine the temporal variation of the value of its Shannon entropy in each case. Our model suggests that the sustained activation of the FGFR communication channel with a step of FGF > 1 nM is required for cell cycle progression and that during the G1/S transition the amount of uncertainty of the GRN remains at a steady value of [~] 2.75 bits, indicating that while the fibroblast stimulation with FGF continues the G1/S transition does not require an additional interchange of information between the emitter and the gene regulatory network to be completed. We also found that either low frequency pulses of FGF or low frequency noise, both with a frequency f [≤] 2.77 Hz, are not filtered by the MAPK cascade and can modify the output of the communication channel, i.e., the amount of the effector proteins c-myc, cdc25A and DUSP. An additional effect suggested by our model is that o low frequency periodic signals and noise possibly blockage cell cycle progression because the threshold value concentration of cdc25A for the G1/S transition is not sustained in the in the nucleus during the 10 hours that this process lasts. Finally, from our model we can estimate the capacity of this communication channel in 0.96 bits/min.


title: Loss of ACKR4 in tumor cells dysregulates dendritic cell migration to tumor-draining lymph nodes and T-cell priming

abstract:
Colorectal cancer (CRC) is one of the most common malignancies in both morbidity and mortality. Immune checkpoint blockade (ICB) treatments have been successful in a portion of mismatch repair-deficient (dMMR) CRC patients but failed in the mismatch repair-proficient (pMMR) CRC patients. Atypical Chemokine Receptor 4 (ACKR4) is known for regulating dendritic cell (DC) migration. However, the roles of ACKR4 in CRC development and immunoregulation are unclear. By analyzing human CRC tissues, transgenic animals, and genetically modified CRC cells lines, our study revealed an important function of ACKR4 in maintaining CRC immune response. Loss of ACKR4 in CRC is associated with poor immune infiltration in the tumor microenvironment. More importantly, loss of ACKR4 in CRC tumor cells, rather than stromal cells, restrains the DC migration and antigen presentation to the tumor-draining lymph nodes (TdLNs). Tumors with ACKR4 knockdown become less sensitive to immune checkpoint blockades. Finally, we identified that microRNA-552 negatively regulates ACKR4 expression in human CRC. Taken together, our work identifies a critical mechanism for the maintenance of the DC-mediated T-cell priming in the TdLNs. These new findings demonstrate a novel mechanism leading to immunosuppression and ICB treatment resistance in CRC tumors.

LAY SUMMARYOur study demonstrated that Atypical Chemokine Receptor 4 (ACKR4) is downregulated in human colorectal cancer (CRC) tissues compared with normal colon tissues. Loss of ACKR4 in human CRC tissues is associated with a weak anti-tumor immune response. Knockdown of ACKR4 in tumor cells impairs the dendritic cell migration from the tumor tissue to the tumor-draining lymph nodes (TdLNs), causing inadequate tumor-specific T-cell expanding and insensitivity to immune checkpoint blockades. However, loss of ACKR4 in tumor stromal cells does not significantly affect anti-tumor immunity. In human CRC tumors, high expression of microRNA-552 is a mechanism leading to ACKR4 downregulation. Our study revealed a novel mechanism that leads to the poor immune response in a subset of CRC tumors and will contribute to the framework for identifying new therapies against this deadly tumor.


title: Engineering a dynamic, controllable infectivity switch in bacteriophage T7

abstract: Transcriptional repressors play an important role in regulating phage genomes. Here, we examined how synthetic regulation based on repressors can be to create a dynamic, controllable infectivity switch in bacteriophage T7. We engineered T7 by replacing a large region of the early phage genome with combinations of ligand-responsive promoters and ribosome binding sites (RBS) designed to control the phage RNA polymerase. Phages with the engineered switch showed virulence comparable to wildtype when not repressed, indicating the phage can be engineered without a loss of fitness. When repressed, the most effective switch used a TetR promoter and a weak RBS, resulting in a two-fold increase in latent period (time to lyse host) and change in phage titer. Further, phage activity could be tuned by varying inducer concentrations. Our study provides a proof of concept for a simple circuit for user control over phage infectivity.

category: synthetic biology


title: Spectral fiber-photometry derives hemoglobin-absorption changes for accurate measurement of fluorescent sensors

abstract:
Fiber-photometry is an emerging technique for recording fluorescent sensor activity in the brain. However, significant hemoglobin-absorption artifacts in fiber-photometry data may be misinterpreted as sensor activity changes. Because hemoglobin exists in nearly every location in the brain and its concentration varies over time, such artifacts could impede the accuracy of many photometry recording results. Here we present a novel use of spectral photometry technique and propose computational methods to quantify photon absorption effects by using activity-independent fluorescence signals, which can be used to derive oxy- and deoxy-hemoglobin concentration changes. Following time-locked neuronal activation in vivo, we observed that a 20% increase in CBV contributes to about a 4% decrease in green fluorescence signal and a 2% decrease in red fluorescence signal. While these hemoglobin concentration changes are often temporally delayed than the fast-responding fluorescence spikes, we found that erroneous interpretation may occur when examining pharmacology-induced sustained activity changes, and in some cases, hemoglobin-absorption could flip the GCaMP signal polarity. We provided hemoglobin-based correction methods to restore fluorescence signals across spectra and compare our results against the commonly used regression approach. We also demonstrated the utility of spectral fiber-photometry for delineating brain regional differences in hemodynamic response functions.

HighlightsO_LIHemoglobin-absorption compromises fiber-photometry recording in vivo
C_LIO_LISpectral photometry allows quantification of hemoglobin concentration changes for correction
C_LIO_LIThe proposed platform allows measuring regional differences in neurovascular transfer function
C_LI


title: Spectral fiber-photometry derives hemoglobin-absorption changes for accurate measurement of fluorescent sensor activity


title: Spectral fiber-photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity


title: FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

abstract: The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of [~] 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the [~] 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZs treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.


title: Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption

abstract: COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.


title: Wolbachia in scale insects: a distinct pattern of infection frequencies and potential transfer routes via ant associates

abstract:
Wolbachia is one of the most successful endosymbiotic bacteria of arthropods. Known as the "master of manipulation", Wolbachia can induce a wide range of phenotypes in its host that can have far-reaching ecological and evolutionary consequences and may be exploited for disease and pest control. However, our knowledge of Wolbachias distribution and infection rate is unevenly distributed across arthropod groups such as scale insects. We fitted a distribution of within-species prevalence of Wolbachia to our data and compared it to distributions fitted to an up-to-date dataset compiled from surveys across all arthropods. The estimated distribution parameters indicate a Wolbachia infection frequency of 43.6% (at a 10% prevalence threshold) in scale insects. Prevalence of Wolbachia in scale insects follows a distribution similar to exponential decline (most species are predicted to have low prevalence infections), in contrast to the U-shaped distribution estimated for other taxa (most species have a very low or very high prevalence). We observed no significant associations between Wolbachia infection and scale insect traits. Finally, we screened for Wolbachia in scale insects ecological associates. We found a positive correlation between Wolbachia infection in scale insects and their ant associates, pointing to a possible route of horizontal transfer of Wolbachia.

Originality-Significance StatementBy creating metadata of Wolbachia infection in arthropods and applying a fitting an advanced mathematical model on the estimated infection frequency in scale insects, a unique pattern of infection prevalence was detected. In addition, ant-scale insect trophallaxis interaction was suggested as a plausible route of Wolbachia transfer


title: Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display

abstract: Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.


title: Force-induced changes of PilY1 drive surface sensing by Pseudomonas aeruginosa

abstract:
During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact is still poorly understood, although mechanosensing is often invoked with little data supporting this claim. Using a combination of molecular genetics and single cell analysis, with biophysical, biochemical and genomics techniques we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain results in modestly lower surface adhesion forces, increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate TFP function in twitching from surface sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains, and its absence in the ~720 sequenced strains of P. aeruginosa PAO1, could contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1.

ImportanceMost bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade are unclear. Here, we provide a key insight into this question: we show that the eukaryotic-like, vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that could account for differing surface colonization strategies.


title: MyoView: Fully-automated image segmentation method to analyse skeletal muscle cross section in exercise-induced regenerating myofibers

abstract: Skeletal muscle is an adaptive tissue with the ability to regenerate in response to exercise training. Cross-sectional area (CSA) quantification, as a main parameter to assess muscle regeneration capability, is highly tedious and time-consuming, necessitating an accurate and automated approach to analysis. Although several excellent programs are available to automate analysis of muscle histology, they fail to efficiently and accurately measure CSA in regenerating myofibers in response to exercise training. Here, we have developed a novel fully-automated image segmentation method based on neutrosophic set algorithms to analyse whole skeletal muscle cross sections in exercise-induced regenerating myofibers, referred as MyoView, designed to obtain accurate fiber size and distribution measurements. MyoView provides relatively efficient, accurate, and reliable measurements for detecting different myofibers and CSA quantification in response to the post-exercise regenerating process. We showed that MyoView is comparable with manual quantification. We also showed that MyoView is more accurate and efficient to measure CSA in post-exercise regenerating myofibers as compared with Open-CSAM, MuscleJ, SMASH and MyoVision. Furthermore, we demonstrated that to obtain an accurate CSA quantification of exercise-induced regenerating myofibers, whole muscle cross-section analysis is an essential part, especially for the measurement of different fiber-types. We present MyoView as a new tool for CSA quantification in skeletal muscle from any experimental condition including exercise-induced regenerating myofibers.


title: New insights into the evolution of SPX gene family from algae to legumes; a focus on soybean

abstract:
BackgroundSPX-containing proteins have been known as key players in phosphate signaling and homeostasis. In Arabidopsis and rice, functions of some SPXs have been characterized, but little is known about their function in other plants, especially in the legumes.

ResultsWe analyzed SPX gene family evolution in legumes and in a number of key species from algae to angiosperms. We found that SPX harboring proteins showed fluctuations in domain fusions from algae to the angiosperms with, finally, four classes appearing and being retained in the land plants. Despite these fluctuations, Lysine Surface Cluster (KSC), and the third residue of Phosphate Binding Sites (PBS) showed complete conservation in almost all of SPXs except few proteins in Selaginella moellendorffii and Papaver sumniferum, suggesting they might have different ligand preferences. In addition, we found that the WGD/segmentally or dispersed duplication types were the most frequent contributors to the SPX expansion, and that there is a positive correlation between the amount of WGD contribution to the SPX expansion in individual species and its number of EXS genes. We could also reveal that except SPX class genes, other classes lost the collinearity relationships among Arabidopsis and legume genomes. The sub- or neo-functionalization of the duplicated genes in the legumes makes it difficult to find the functional orthologous genes. Therefore, we used two different methods to identify functional orthologs in soybean and Medicago. High variance in the dynamic and spatial expression pattern of GmSPXs proved the new or sub-functionalization in the paralogs.

ConclusionThis comprehensive analysis revealed how SPX gene family evolved from algae to legumes and also discovered several new domains fused to SPX domain in algae. In addition, we hypothesized that there different phosphate sensing mechanisms might occur in S. moellendorffii and P. sumniferum. Finally, we predicted putative functional orthologs of AtSPXs in the legumes, especially, orthologs of AtPHO1 and AtPHO1;H1, involved in long-distance Pi transportation. These findings help to understand evolution of phosphate signaling and might underpin development of new legume varieties with improved phosphate use efficiency.

category: evolutionary biology


title: A gene-based capture assay for surveying patterns of genetic diversity and insecticide resistance in a worldwide group of invasive mosquitoes

abstract: Understanding patterns of diversification, genetic exchange, and pesticide resistance in insect species of human health concern is necessary for effective population reduction and management. With the broad availability of next-generation sequencing technologies, one of the best approaches for surveying such patterns involves the simultaneous genotyping of many samples for large numbers of genetic markers from across the known genome. To this end, the targeting of gene sequences of known function or inheritance can be a cost-effective strategy. One insect group of substantial health concern are the mosquito taxa that make up the Culex pipiens complex. Members of this complex transmit damaging arboviruses and filariae worms to humans, as well as other pathogens that are detrimental to endangered vertebrate species such as bird malaria. Here we describe our development of a targeted gene-based assay for surveying genetic diversity and population structure in this mosquito complex. To test the utility of this assay, we examined taxonomic divergence among samples from several members of the complex, as well as distinct populations of the relatively under-studied Culex quinquefasciatus, an urban pantropical species. We also examined the presence of known insecticide-resistance conferring alleles. Broadly, our developed gene-based assay proved effective for examining patterns of taxonomic and geographic clustering within the species complex, as well as for surveying genetic variants that have been associated with insecticide resistance. This assay will be useful for future studies that aim to understand the genetic mechanisms underlying the evolution of ubiquitous and increasingly damaging disease vectors.

category: genomics


title: Gene Expression Risk Scores for COVID-19 Illness Severity

abstract:
BackgroundThe correlates of COVID-19 illness severity following infection with SARS-Coronavirus 2 (SARS-CoV-2) are incompletely understood.

MethodsWe assessed peripheral blood gene expression in 53 adults with confirmed SARS-CoV-2-infection clinically adjudicated as having mild, moderate or severe disease. Supervised principal components analysis was used to build a weighted gene expression risk score (WGERS) to discriminate between severe and non-severe COVID.

ResultsGene expression patterns in participants with mild and moderate illness were similar, but significantly different from severe illness. When comparing severe versus non-severe illness, we identified >4000 genes differentially expressed (FDR<0.05). Biological pathways increased in severe COVID-19 were associated with platelet activation and coagulation, and those significantly decreased with T cell signaling and differentiation. A WGERS based on 18 genes distinguished severe illness in our training cohort (cross-validated ROC-AUC=0.98), and need for intensive care in an independent cohort (ROC-AUC=0.85). Dichotomizing the WGERS yielded 100% sensitivity and 85% specificity for classifying severe illness in our training cohort, and 84% sensitivity and 74% specificity for defining the need for intensive care in the validation cohort.

ConclusionThese data suggest that gene expression classifiers may provide clinical utility as predictors of COVID-19 illness severity.


title: MEOX2 homeobox gene promotes growth of malignant gliomas

abstract:
Glioblastoma (GBM) is an aggressive tumor that frequently exhibits gain of chromosome 7, loss of chromosome 10 and aberrantly activated receptor tyrosine kinase signaling pathways. Here, we identify mesenchyme homeobox 2 (MEOX2) on chromosome 7 with increased expression in GBM as a salient oncogenic transcription factor. Specifically, we show that MEOX2 overexpression leads to increased ERK phosphorylation, and we identify a phosphorylation site on MEOX2 that regulates its transcriptional activity by altering its subnuclear localization. We show that MEOX2 overexpression can lead to increased growth in GBM implantation models and cooperates with loss of p53 and PTEN in cerebral organoid models of human malignant gliomas to induce cell proliferation. Furthermore, using high-throughput genomics, we identify transcriptional target genes of MEOX2 in patient-derived GBM tumorsphere models and a fresh frozen GBM tumor. These analyses show that MEOX2 activates several oncogenic pathways involved in MAPK signaling and extracellular matrix organization. Furthermore, MEOX2 binds to oncogenic ETS factors and known glioma oncogenes such as FABP7. In total, we reveal a novel role for MEOX2 in GBM initiation and progression and demonstrate that MEOX2 can enhance ERK signaling through a feed-forward mechanism.

Significance StatementGlioblastoma (GBM) harbors gain of chromosome 7 as an early driver event. In this study, we show that mesenchyme homeobox 2 (MEOX2), an aberrantly upregulated transcription factor on chromosome 7, is an oncogene in human glioblastoma. In contrast to GBM, MEOX2 expression is very low in normal brain. We show that MEOX2 cooperates with p53 and PTEN loss to promote tumor initiation in cerebral organoid models. In addition, we identify direct and indirect molecular targets of MEOX2 and demonstrate its role in activating the ERK signaling cascade. These findings identify a novel oncogene in GBM and highlight the transcriptional networks hijacked by these tumors to activate signaling pathways central to GBM biology.


title: CULTURE AND MAINTENANCE OF URINE-DERIVED, 3-DIMENSIONAL CANINE TRANSITIONAL CELL CARCINOMA ORGANOIDS

abstract: Bladder cancer is the ninth most common malignancy in the world. Transitional cell carcinoma (TCC), also referred to as urothelial carcinoma (UC) is the most common form of bladder cancer, occurring in 90% of cases. In this study, we explore urine-derived, 3-dimensional, canine TCC organoids as a possible model to study bladder TCC ex vivo. After establishing the cell lines, we subjected the 3D cells to RNA in situ hybridization (RNA-ISH) and cell viability assays. Overall, 3D cell culture from urine samples of TCC diagnosed canines expressed RNA biomarkers in a similar manner to parent tumors via RNA-ISH and showed more sensitivity to cisplatin treatment when compared to 2D human TCC cells. With further experimentation, canine TCC organoids could become an ideal model to study TCC ex vivo.


title: A modular platform for on-demand vaccine self-assembly enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

abstract: Engineered outer membrane vesicles (OMVs) derived from laboratory strains of bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. As mimics of the bacterial cell surface, OMVs offer a molecularly-defined architecture for programming repetitive, high-density display of heterologous antigens in conformations that elicit strong B and T cell immune responses. However, antigen display on the surface of OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. To address this shortcoming, we created a universal approach called AddVax (avidin-based dock- and-display for vaccine antigen cross (x)-linking) whereby virtually any antigen that is amenable to biotinylation can be linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen receptor (SNARE) comprised of an outer membrane scaffold protein fused to a member of the avidin family. We show that SNARE-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations were injected in wild-type BALB/c mice, strong antigen-specific antibody responses were observed that depended on the physical coupling between the antigen and SNARE-OMV delivery vehicle. Overall, these results demonstrate AddVax as a modular platform for rapid self-assembly of antigen-studded OMVs with the potential to accelerate vaccine generation, respond rapidly to pathogen threats in humans and animals, and simplify vaccine stockpiling.


title: Immunization with SARS-CoV-2 nucleocapsid protein triggers a pulmonary immune response in rats

abstract: The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

category: immunology


title: A base-line cellular antiviral state is maintained by cGAS and its most frequent naturally occurring variant rs610913

abstract: Upon recognition of aberrantly located DNA, the innate immune sensor cGAS activates STING/IRF-3-driven antiviral responses. Here we characterized the ability of a specific variant of the cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted towards the possibility for an additional binding site for a potential cellular co-factor in cGAS dimers. However, cGAS(WT) or cGAS(P261H)-reconstituted THP-1 cGAS KO cells shared steady-state expression of interferon-stimulated genes (ISGs), as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared to cGAS KO- or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared to cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend towards a slightly reduced type I IFN response as compared to PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a base-line antiviral state rendering cells more refractory to ISG-sensitive viral infections. Even though rs610913 failed to grossly differ phenotypically from the wild-type gene, its expression potentially results in a slightly altered susceptibility to viral infections in vivo.


title: The AIRE G228W mutation results in a longer-lasting AIRE-SIRT1 interaction

abstract:
The in silico and in vitro binding of a peptide covering a part of the autoimmune regulator (AIRE) SAND domain with the SIRT1 protein provides a powerful model system for studying the mechanism of the dominant SAND G228W mutation, which is the causative of APS-1 autoimmune syndrome. It is known that the mutant G228W AIRE protein accumulates more within the nucleus of cells than its wild-type counterpart does. This accumulation is not physiological and is associated with loss of AIRE function. However, the precise molecular mechanism that leads to AIRE accumulation is not yet known. AIRE works as a tetramer and interacts with partner proteins to form the "AIRE complex" that pushes RNA Pol II stalling in the chromatin of medullary thymic epithelial cells. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates Lys residues of the SAND domain. Once AIRE is deacetylated, the binding with SIRT1 is undone, allowing the complex to proceed downstream. Here, we integrate molecular modeling, docking, dynamics, and surface plasmon resonance approaches to compare the structure and energetics of binding/release between AIRE G228 (wild-type) or W228 (mutant) peptides to SIRT1. We find that the proximity of G228W mutation to a K aminoacid residue in the SAND domain promotes a longer-lasting AIRE-SIRT1 interaction. The lasting interaction might cause a delay in the AIRE SAND domain to be released from the SIRT1 catalytic site, which might cause accumulation of the defective AIRE mutant protein in the nuclei of cells.

SignificanceThis report reveals the mechanism of the pathogenic and dominant G228W mutation in the AIRE SAND domain. The G228W mutation is found in APS-1 syndrome patients, and it is critical to understand the molecular basis of loss of self-representation, a challenging aspect for immunology. Through modeling, molecular dynamics, and protein binding kinetics, we found that the G228W mutation leads to a stronger physical interaction between the AIRE SAND domain and the SIRT1 protein when compared to the equivalent wild-type segment. The short-term consequence of this stronger interaction is that the release of the AIRE-SIRT1 binding is slower. This might explain the reason that cells carrying the G228W mutation accumulate AIRE protein in their nuclei. This finding reveals with precision the AIRE-SIRT1 binding and the molecular mechanism of the human AIRE G228W mutation.


title: Key Drivers of Ecological Assembly in the Hindgut of Atlantic Cod (Gadus morhua) when Fed with a Macroalgal Supplemented diet - How Robust Is the Gut to Taxonomic Perturbation?

abstract: The microbiota in the gastrointestinal tract of any species is shaped by internal and external cues in addition to random events which can be difficult to disentangle from a range of interacting variables. Estimating ecological assembly processes can help elucidate these factors. In our study, farmed Atlantic cod (Gadus morhua) were fed a diet of 10% macroalgae supplement (Ulva rigida species [ULVA] or Ascophyllum nodosum [ASCO] or a non-amended control diet [CTRL]) over a 12-week period and the ecological assembly processes quantified. The CTRL samples showed selection (variable selection - divergence in communities related to selective environmental conditions) as a key assembly process, while dispersal limitation (limited movement results in divergent communities through stochastic processes) was a driver of the gut microbiome for fish fed the macroalgae supplemented diet at Week 12 (i.e., ASCO and ULVA). Fish from the ASCO grouping diverged into ASCO_N (normal) and ASCO_LG (lower growth), where ASCO_LG individuals found the diet unpalatable. The recruitment of new taxa over time was altered in the ASCO_LG fish, with the gut microbiome showing phylogenetic under dispersion (nepotistic recruitment of species). Finally, the gut microbiome (CTRL and ULVA only) showed increasing robustness to taxonomic disturbance over time and an increase in functional redundancy. This study advances our understanding of the ecological assembly and succession in the hindgut of juvenile Atlantic cod across dietary regimes. Understanding the processes driving ecological assembly in the gut microbiome, in fish research specifically, could allow us to manipulate the microbiome for improved health or resilience to disease for improved aquaculture production.


title: Species-scale genomic analysis of S. aureus genes influencing phage host range and their relationships to virulence and antibiotic resistance genes

abstract:
Phage therapy has been proposed as a possible alternative treatment for infections caused by the ubiquitous bacterial pathogen Staphylococcus aureus. However, successful phage therapy requires knowing both host and phage genetic factors influencing host range for rational cocktail formulation. To further our understanding of host range, we searched 40,000+ public S. aureus genome sequences for previously identified phage resistance genes. We found that phage adsorption targets and genes that block phage assembly were significantly more conserved than genes targeting phage biosynthesis. Core phage resistance genes had similar nucleotide diversity, ratio of non-synonymous to synonymous substitutions, and functionality (measured by delta-bitscore) to other core genes in a set of 380 non-redundant S. aureus genomes (each from a different MLST sequence type). Non-core phage resistance genes were significantly less consistent with the core genome phylogeny than all non-core genes in this set. Only superinfection immunity genes correlated with empirically determined temperate phage resistance, accessory genome content, and numbers of accessory antibiotic resistance or virulence genes encoded per strain. Taken together, these results suggested that, while phage adsorption genes are heavily conserved in the S. aureus species, they are not undergoing positive selection, arms race dynamics. They also suggested genes classified as involved in assembly are least phylogenetically constrained and superinfection immunity genes best predict both empirical phage resistance and levels of phage-mediated HGT.

ImportanceStaphylococcus aureus is a widespread, hospital- and community-acquired pathogen that is commonly antibiotic resistant. It causes diverse diseases affecting both the skin and internal organs. Its ubiquity, antibiotic resistance, and disease burden make new therapies urgent, such as phage therapy, in which viruses specific to infecting bacteria clear infection. S. aureus phage host range not only determines whether phage therapy will be successful by killing bacteria but also horizontal gene transfer through transduction of host genetic material by phages. In this work, we comprehensively reviewed existing literature to build a list of S. aureus phage resistance genes and searched our database of almost 43,000 S. aureus genomes for these genes to understand their patterns of evolution, finding that prophages superinfection immunity correlates best with phage resistance and HGT. These findings improved our understanding of the relationship between known phage resistance genes and phage host range in the species.


title: Detection and phylogeny of Wolbachia in field-collected Aedes albopictus and Aedes aegypti from Manila City, Philippines

abstract:
BackgroundWolbachia is the most common bacterial endosymbiont of arthropods, such as the medically important Aedes albopictus and recent reports also detected in Aedes aegypti. Our study showed additional support for the presence of wolbachia in Ae. albopictus and Ae. aegypti using Wolbachia specific markers, wsp, and 16S.

Main textThis study collected 12 adult Ae. albopictus and 359 Ae. aegypti from 183 households in a dengue-prone area, Manila, Philippines, between June and September 2017. Aedes larvae (n = 509) were also collected from 17 water containers from 11 households. The DNA of the Aedes larvae and adults were screened for the presence of Wolbachia using the wsp and 16S markers, following optimized polymerase chain reaction (PCR) conditions, and sequenced. Our results showed that 3 out of 359 (0.84%) adult Ae. aegypti and 12 out of 12 (100%) adult Ae. albopictus were Wolbachia positive, whereas all larvae tested negative for Wolbachia (0/509; 0%). The wsp marker revealed six Wolbachia-positive Ae. albopictus, whereas the 16S marker showed Wolbachia in three Ae. aegypti and ten Ae. albopictus

ConclusionOur results suggest that the utilization of two Wolbachia specific markers, wsp and 16S demonstrated Wolbachia presence in individual Ae. albopictus and Ae. aegypti. The results of the Wolbachia infection in Ae. albopictus showed that the detected strains were from either supergroups A and B. Despite the low infection rate of Wolbachia in Ae. aegypti, our results demonstrated the presence of Wolbachia in field-collected Ae. aegypti supporting previous studies.


title: Host ecology regulates interspecies recombination in bacteria of the genus Campylobacter

abstract: Horizontal gene transfer (HGT) can allow traits that have evolved in one bacterial species to transfer to another. This has potential to rapidly promote new adaptive trajectories such as zoonotic transfer or antimicrobial resistance. However, for this to occur requires gaps to align in barriers to recombination within a given time frame. Chief among these barriers is the physical separation of species with distinct ecologies in separate niches. Within the genus Campylobacter there are species with divergent ecologies, from rarely isolated single host specialists to multi-host generalist species that are among the most common global causes of human bacterial gastroenteritis. Here, by characterising these contrasting ecologies, we can quantify HGT among sympatric and allopatric species in natural populations. Analysing recipient and donor population ancestry among genomes from 30 Campylobacter species we show that cohabitation in the same host can lead to a 6-fold increase in HGT between species. This accounts for up to 30% of all SNPs within a given species and identifies highly recombinogenic genes with functions including host adaptation and antimicrobial resistance. As described in some animal and plant species, ecological factors are a major evolutionary force for speciation in bacteria and changes to the host landscape can promote partial convergence of distinct species through HGT.


title: High Definition DIC Imaging Uncovers Transient Stages of Pathogen Infection Cycles on the Surface of Human Adult Stem Cell-Derived Intestinal Epithelium

abstract:
Interactions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study e.g. microbe motility, binding and invasion of host cells, and intra-host-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging, but poorly reflects the host tissues physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC monolayers are grown in alumina membrane chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows sub-second visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica Typhimurium (Salmonella) and Giardia intestinalis (Giardia), at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium, showing that Salmonella adheres to the IEC surface for prolonged periods before host-cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. This method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. Thereby, this work describes a powerful, generalizable, and relatively inexpensive approach to study dynamic pathogen interactions with IEC surfaces at high resolution and under near-native conditions.

ImportanceKnowledge of dynamic niche-specific interactions between single microbes and host cells is essential to understand infectious disease progression. However, advances in this field have been hampered by the inherent conflict between the technical requirements for high resolution live-cell imaging on one hand, and conditions that best mimic physiological infection niche parameters on the other. Towards bridging this divide, we present methodology for differential interference contrast (DIC) imaging of pathogen interactions at the apical surface of enteroid-derived intestinal epithelia, providing both high spatial and temporal resolution. This alleviates the need for fluorescent reporters in live-cell imaging and provides dynamic information about microbe interactions with a non-transformed, confluent, polarized and microvilliated human gut epithelium. Using this methodology, we uncover previously unrecognized stages of Salmonella and Giardia infection cycles at the epithelial surface.


title: Characterization of the flagellar collar reveals structural plasticity essential for spirochete motility

abstract: Spirochetes are a remarkable group of bacteria with distinct morphology and periplasmic flagella that enable motility in viscous environments, such as host connective tissues. The collar, a spirochete-specific complex of the periplasmic flagellum, is required for the unique spirochete motility, yet it has not been clear how the collar assembles and enables spirochetes to transit between complex host environments. Here, we characterize the collar complex in the Lyme disease spirochete Borrelia burgdorferi. We discover as well as delineate the distinct functions of two novel collar proteins, FlcB and FlcC, by combining subtractive bioinformatic, genetic, and cryo-electron tomography approaches. Our high-resolution in-situ structures reveal that the multi-protein collar has a remarkable structural plasticity essential not only for assembly of flagellar motors in the highly curved membrane of spirochetes but also for generation of the high torque necessary for spirochete motility.


title: Multi-omics investigation of Clostridioides difficile-colonized patients reveals protective commensal carbohydrate metabolism

abstract: Clostridioides difficile infection (CDI) imposes a substantial burden on the health care system in the United States. Understanding the biological basis for the spectrum of C. difficile-related disease manifestations is imperative to improving treatment and prevention of CDI. Here, we investigate the correlates of asymptomatic C. difficile colonization using a multi-omics approach, comparing the fecal microbiome and metabolome profiles of patients with CDI versus asymptomatically-colonized patients. We find that microbiomes of asymptomatic patients are significantly enriched for species in the class Clostridia relative to those of symptomatic patients. Asymptomatic patient microbiomes were enriched with fucose, rhamnose, and sucrose degradation pathways relative to CDI patient microbiomes. Fecal metabolomics corroborates this result: we identify carbohydrate compounds enriched in asymptomatic patients relative to CDI patients, and correlated with a number of commensal Clostridia. Further, we reveal that across C. difficile isolates, the carbohydrates rhamnose and lactulose do not serve as robust growth substrates in vitro, corroborating their enriched detection in our metagenomic and metabolite profiling of asymptomatic individuals. We conclude that in asymptomatically-colonized individuals, carbohydrate metabolism by other commensal Clostridia may prevent CDI by inhibiting C. difficile proliferation. These insights into C. difficile colonization and putative commensal competition suggest novel avenues to develop probiotic or prebiotic therapeutics against CDI.