title: Prediction and validation of the three-dimensional structure of glucokinase-1 from Phytophthora infestans

abstract: According to its primary structure, the [PITG_06016] gene encodes for one of the 7 glucokinases present in Phytophthora infestans (PiGlcK-1), the causal agent of late blight disease. Currently, there are no structural studies of any of its enzymes, being the determination of the three-dimensional (3D) structure of PiGlcK-1 a necessary contribution in the deduction of its functions, its interaction with ligands, and possible regulatory mechanisms. In this work we present the first structural model obtained by in silico tools for PiGlcK-1. For the prediction of this model, different algorithms were used to find the best annealing, refinement, and qualitative evaluation of them. A structural comparison of the predicted model with other structures of crystallized kinase enzymes allowed us to identify the regions of interaction with their classical substrates (glucose and ATP), as well as to identify the amino acid residues involved in the binding of other substrates such as fructose and ADP. In addition, we propose a possible recognition region of PPi, an activator of kinase activity that includes the GXGE motif, conserved in enzymes of the ribokinase (RK) family, which distinguishes this PiGlcK-1 from a classical glucokinase. Accordingly, these findings suggest PPi-binding motif as potential targets for the development of inhibitors of PiGlcK-1 activity.


title: Applying Historical Data in a Nonlinear Mixed-Effects Model Can Reduce the Number of Control Rats Required for Calculation of the Relative Potency of Insulin Analogues

abstract: This paper examines how to reduce the number of control animals in preclinical hyperinsulemic glucose clamp studies if we make use of information on historical studies. A data set consisting of 59 studies in rats to investigate new insulin analogues for diabetics, collected in the years 2000 to 2015, is analysed. A simulation experiment is performed based on a carefully built nonlinear mixed-effects model including historical information, comparing results (for the relative log-potency) with the standard approach ignoring previous studies. We find that by including historical information in the form of the mixed-effects model proposed, we can to remove between 23% and 51% of the control rats in the two studies looked closely upon to get the same level of precision on the relative log-potency as in the standard analysis. How to incorporate the historical information in the form of the mixed-effects model is discussed, where both a meta approach as well as a Bayesian approach are suggested. The conclusions are similar for the two approaches, and therefore, we conclude that the inclusion of historical information is beneficial in regard to using fewer control rats.

category: pharmacology and toxicology


title: Pro-apoptotic caspase deficiency reveals a cell-extrinsic mechanism of NK cell regulation

abstract: AO_SCPLOWBSTRACTC_SCPLOWRegulated cell death is essential for the maintenance of cellular and tissue homeostasis. In the hematopoietic system, genetic defects in apoptotic cell death generally produce the accumulation of immune cells, inflammation and autoimmunity. In contrast, we found that genetic deletion of caspases of the mitochondrial apoptosis pathway reduces natural killer (NK) cell numbers and makes NK cells functionally defective in vivo and in vitro. Caspase deficiency results in constitutive activation of a type I interferon (IFN) response, due to leakage of mitochondrial DNA and activation of the cGAS/STING pathway. The NK cell defect in caspase-deficient mice is independent of the type I IFN response, but the phenotype is partially rescued by cGAS or STING deficiency. Finally, caspase deficiency alters NK cells in a cell-extrinsic manner. Type I IFNs and NK cells are two essential effectors of antiviral immunity, and our results demonstrate that they are both regulated in a caspase-dependent manner. Beyond caspase-deficient animals, our observations may have implications in infections that trigger mitochondrial stress and caspase-dependent cell death.

category: immunology


title: Elongated sample plot shapes produce higher measurement precision in clustering populations

abstract: Monitoring the density of natural populations is crucial for ecosystem management decision making and natural resource management. The most widely used method to measure the population density of animal and plant species in natural habitats is to count organisms in sample plots. Yet evaluation of survey performance by different sample plot shapes, e.g. quadrats compared with transects, has been largely neglected since the 1990s and has not been undertaken using simulation. Simulating populations and surveys, we evaluated population density measurement precision for 900 cases, testing 30 sample plot survey designs in each of 30 spatially clustered populations. We varied three design options: elongation of plot shape while keeping sample area constant, systematic or random plot allocation, and sample size. Survey design performance varied markedly: elongating the plot shape always improved survey precision; allocating plots systematically sometimes did. (i) Averaged across all tested populations, elongated (1:100) transect plot shapes were 2-to-3 times more precise than square (10:10) quadrats. (ii) The precision of systematic surveys accelerated with sample plot number, increasing faster than the (known) linear increase under simple random sampling. This non-linear, concave upward, dependence of systematic precision on sample size has not previously been reported. (iii) The most precise design we evaluated used long narrow transects allocated systematically. Averaging among all 30 tested populations, a researcher would need 600 random square (10:10) quadrats to equal the precision achieved by 100 systematic (1:100) transects. Finding this average efficiency difference of 600% for a survey sample size of 100 plots, these simulation results imply that field trips requiring five sampling days using random quadrats could achieve equal precision in one or two days using systematic elongated transects. For all clustered populations we tested, long narrow transects resulted in a more efficient design for sample plot survey.

category: ecology


title: Another step towards understanding evolutionary changes in Bruggmanniella Tavares, 1909 group (Diptera, Cecidomyiidae, Asphondyliini)

abstract: An update of the delimitation of the genus Bruggmanniella based on phylogenetic analysis using morphological data is presented. We included the seven new species of Bruggmanniella described between 2019 and 2020, and discuss some aspects of the evolutionary changes among the closely related genera Bruggmanniella, Pseudasphondylia, and Illiciomyia. Bruggmanniella is confirmed here as a monophyletic Neotropical lineage, divergent from the Asian species. The phylogenetic reconstruction hypothesized here reinforces the pertinence of the genus Odontokeros to house all species occurring in the Oriental/Palearctic region under Bruggmanniella. The delimitation of Bruggmanniella, the geographical distribution, and niche occupation are discussed.

category: zoology


title: The Ventrolateral Anterior Temporal Lobe is Commonly Engaged by Both Mental State Inference and Semantic Association Tasks

abstract: A key challenge for neurobiological models of social cognition is to elucidate whether brain regions are specialised for that domain. In recent years, discussion surrounding the role of anterior temporal regions epitomises such debates; some argue the anterior temporal lobe (ATL) is part of a domain-specific network for social processing, while others claim it comprises a domain-general hub for semantic representation. In the present study, we used ATL-optimised fMRI to map the contribution of different ATL structures to a variety of paradigms frequently used to probe a crucial social ability, namely  theory of mind (ToM). Using multiple tasks enables a clearer attribution of activation to ToM as opposed to idiosyncratic features of stimuli. Further, we directly explored whether these same structures are also activated by a non-social task probing semantic representations. We revealed that common to all of the tasks was activation of a key ventrolateral ATL region that is often invisible to standard fMRI. This constitutes novel evidence in support of the view that the ventrolateral ATL contributes to social cognition via a domain-general role in semantic processing, and against claims of a specialised social function.

category: neuroscience


title: The Role of the Ventrolateral Anterior Temporal Lobes in Social Cognition


title: The language network supports both lexical access and sentence generation during language production

abstract: A network of left frontal and temporal brain regions has long been implicated in language comprehension and production. However, because of relatively fewer investigations of language production, the precise role of this  language network in production-related cognitive processes remains debated. Across four fMRI experiments using picture naming/description to mimic the translation of conceptual representations into words/sentences, we characterize the response of the language regions to production demands using individual-subject-level analyses. We report three novel results. First, sentence production elicits strong responses in the language network regardless of output modality (speaking vs. typing). Second, the language regions respond to both lexical access and sentence-generation demands, mirroring the strong integration between lexico-semantic and combinatorial processes that has been observed in language comprehension. Finally, we find no evidence of brain regions--including outside the language network--that selectively support sentence generation. Instead, language regions respond overall more strongly during production than during comprehension for similar linguistic strings, suggesting that production incurs a greater cost for the language network. Together, these results align with the idea that language comprehension and production draw on the same knowledge representations, which are stored in the language-selective network and are used both to interpret and generate linguistic utterances.


title: Human-specific enrichment of schizophrenia risk-genes in callosal neurons of the developing neocortex

abstract: Human genetic studies have provided a wealth of information on genetic risk factors associated with neuropsychiatric diseases. However, whether different brain cell types are differentially affected in disease states and when in their development and maturation alterations occur is still poorly understood. Here we generated a longitudinal transcriptional map of excitatory projection neuron (PN) and inhibitory interneuron (IN) subtypes of the cerebral cortex, across a timeline of mouse embryonic and postnatal development, as well as fetal human cortex and human cortical organoids. We found that three types of gene signatures uniquely defined each cortical neuronal subtype: dynamic (developmental), adult (terminal), and constitutive (stable), with individual neuronal subtypes varying in the degree of similarity of their signatures between species. In particular, human callosal projection neurons (CPN) displayed the greatest species divergence, with molecular signatures highly enriched for non-coding, human-specific RNAs. Evaluating the association of neuronal class-specific signatures with neuropsychiatric disease risk genes using linkage disequilibrium score regression showed that schizophrenia risk genes were enriched in CPN identity signatures from human but not mouse cortex. Human cortical organoids confirmed the association with excitatory projection neurons. The data indicate that risk gene enrichment is both species- and cell type-specific. Our study reveals molecular determinants of cortical neuron diversification and identifies human callosal projection neurons as the most species-divergent population and a potentially vulnerable neuronal class in schizophrenia.

category: developmental biology


title: Cysteine induces mitochondrial reductive stress in glioblastoma through hydrogen peroxide production

abstract: Glucose and amino acid metabolism are critical for glioblastoma (GBM) growth, but little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated The Cancer Genome Atlas for alterations in glucose and amino acid signatures in GBM relative to other human cancers and found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers. Treatment of patient-derived GBM cells with the FDA-approved cysteine compound N-acetylcysteine (NAC) reduce GBM cell growth and mitochondrial oxygen consumption, which was worsened by glucose starvation. Mechanistic experiments revealed that cysteine compounds induce rapid mitochondrial H2O2 production and reductive stress in GBM cells, an effect blocked by oxidized glutathione, thioredoxin, and redox enzyme overexpression. These findings indicate that GBM is uniquely susceptible to NAC-driven reductive stress and could synergize with glucose-lowering treatments for GBM.

category: cancer biology


title: Combining CRISPR/Cas9 and brain imaging: from genes to proteins to networks

abstract: Receptors, transporters and ion channels are important targets for therapy development in neurological diseases including Alzheime[r]s disease, Parkinso[n]s disease, epilepsy, schizophrenia and major depression. Several receptors and ion channels identified by next generation sequencing may be involved in disease initiation and progression but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on whole brain level. Here, we combine CRISPR/Cas9 gene-editing with in vivo positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene networks relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR/Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a novel tool to investigate the impact of specific genes on brain molecular and functional dynamics which will help to develop tailored therapies for normalizing brain function. The method can easily be transferred to higher-order species allowing for a direct comparison of the molecular imaging findings.


title: Combining CRISPR/Cas9 and brain imaging: from genes to molecules to networks


title: Genetic variations within human gained enhancer elements affect human brain sulcal morphology.

abstract: The expansion of the cerebral cortex is one of the most distinctive changes in the evolution of the human brain. Cortical expansion and related increases in cortical folding may have contributed to emergence of our capacities for high-order cognitive abilities. Molecular analysis of humans, archaic hominins, and non-human primates has allowed identification of chromosomal regions showing evolutionary changes at different points of our phylogenetic history. In this study, we assessed the contributions of genomic annotations spanning 30 million years to human sulcal morphology measured via MRI in more than 18,000 participants from the UK Biobank. We found that variation within brain-expressed human gained enhancers, regulatory genetic elements that emerged since our last common ancestor with Old World monkeys, explained more trait heritability than expected for the left and right calloso-marginal posterior fissures and the right central sulcus. Intriguingly, these are sulci that have been previously linked to the evolution of locomotion in primates and later on bipedalism in our hominin ancestors.


title: Population differentiation of polygenic score predictions under stabilizing selection

abstract: 1Given the many small-effect loci uncovered by genome-wide association studies (GWAS), polygenic scores have become central to the drive for genomic medicine and have spread into various areas including evolutionary studies of adaptation. While promising, these scores are fraught with issues of portability across populations, due to mis-estimated effect sizes and missing causal loci across populations unrepresented in large-scale GWAS. The poor portability of polygenic scores at first seems at odds with the view that much of common genetic variation is shared among populations. Here we investigate one potential cause of this discrepancy, stabilizing selection on complex traits. Somewhat counter-intuitively, while stabilizing selection to the same optimum phenotype leads to lower phenotypic differentiation among populations, it increases genetic differentiation at GWAS loci because it accelerates the turnover of polymorphisms underlying trait variation within populations. We develop theory to show how stabilizing selection impacts the utility of polygenic scores when applied to unrepresented populations. Specifically, we quantify their reduced prediction accuracy and find they can substantially overstate average genetic differences of phenotypes among populations. Our work emphasizes stabilizing selection to the same optimum as a useful null evolutionary model to draw connections between patterns of allele frequency and polygenic score differentiation.

category: evolutionary biology


title: LRP-1 Commands Two Parallel Signalling Mechanisms to Support Constitutive Tumour Cell Motility in the Absence of Blood Supply

abstract: Tumour cells often face the stress of ischemic (nutrient paucity and hypoxia) environment and must act self-sufficient to migrate toward the nearest blood supply or die. The mechanism that supports the constitutive motility of tumour cells under stress is poorly understood. We and others have previously shown that the low-density lipoprotein receptor-related protein 1 (LRP-1) plays a critical role in tumour cell migration and invasion in vitro and tumour formation in mice. Herein we show that depletion of LRP-1 completely abolishes the self-supported and serum-independent tumour cell motility. More intriguingly, we demonstrate that LRP-1 commands the full tumour cell motility by connecting with two independent cell surface signalling pathways. First, LRP-1 mediates secreted Hsp90 signalling via the "Hsp90 > LRP-1 receptor autocrine loop" for a half of tumour cell motility. Second, LRP1 stabilizes constitutively activated EGFR signalling that contributes the other half of tumour cell motility. Only combined inhibitions of the secreted Hsp90 autocrine and the EGFR signalling reproduces the effect of LRP1 down-regulation on constitutive tumour cell motility. This study reveals a novel mechanism of how tumour cells migrate in the absence of blood support.

category: cell biology


title: Generation and diversification of recombinant monoclonal antibodies for studying mitosis

abstract: Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitotic cell division; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.


title: Mechanical Causation of Biological Structure: Productive Pulls Produce Persistent Filaments in a Human Fibroblast Model of Matrix Development

abstract: During development, mesenchymal cells direct the elaboration of extracellular matrix that shapes the initial animal Bauplan which subsequently grows to produce mechanically-competent structure. To gain insight into the processes that initiate matrix formation at the cellular level, high temporal and spatial resolution videos were obtained from a primary human corneal fibroblast (PHCF) cell culture system known to produce an organized, collagenous stroma similar to a human cornea. The videos were taken over a 4-day period as the culture was approaching confluency, which permitted a clear view of cell kinematics and any elaborated filaments. The movies reveal an active cellular system in which the PHCFs execute five types of high-velocity and high extensional strain-rate "pulls" that produce persistent filaments. In four of the pull types, average maximum strain rates ([~]0.1-0.33 s-1) were adequate to induce aggregation and/or crystallization in crowded biopolymer systems. The results demonstrate that PHCFs have the capacity to mechanically induce the formation of biopolymer structures intercellularly and in the path of force.


title: Unique features of transcription termination and initiation at closely spaced tandem human genes

abstract: The synthesis of RNA Polymerase II (Pol2) products, which include messenger RNAs or long noncoding RNAs, culminates in transcription termination. How the transcriptional termination of a gene impacts the activity of promoters found immediately downstream of it, and which can be subject to potential transcriptional interference, remains largely unknown. We examined in an unbiased manner features of the intergenic region of pairs of tandem and closely spaced (<2kb) genes found on the same strand. Intergenic regions separating tandem genes are enriched with Guanines and are characterized by binding of several proteins, including AGO1 and AGO2 of the RNA interference pathway. Additionally, we found that Pol2 with a specific modification pattern is particularly enriched in this region, and it is lost upon perturbations affecting splicing or transcriptional elongation. Perturbations of genes involved in Pol2 pausing and R loop biology preferentially affect expression of downstream genes in tandem gene pairs. Overall, we find that features associated with potential Pol2 recycling rather than those associated with avoidance of transcriptional interference are the predominant driving force shaping these regions.

category: genomics


title: CAMOIP: A Web Server for Comprehensive Analysis on Multi-Omics of Immunotherapy in Pan-cancer

abstract: Immune checkpoint inhibitors (ICIs) have completely changed the therapeutic approach for tumor patients. Immunotherapy has also produced much needed data about mutation, expression, and prognosis, providing an unprecedented opportunity for discovering candidate drug targets and screening for immunotherapy-relevant biomarkers. Although existing web tools enable biologists to analyze the expression, mutation, and prognosis data on tumors, they are currently not able to carry out data mining and mechanism analyses related to immune checkpoint therapy. Thus, we developed our own web-based tool called Comprehensive Analysis on Multi-Omics of Immunotherapy in Pan-cancer (CAMOIP), in which we can screen prognostic markers and analyze the mechanisms involved with markers and immunotherapy (more than 4000 patients). The analyses include survival analysis, expression analysis, drug sensitivity analysis, mutational landscape, immune checkpoint analysis, immune related signature analysis, immune cell analysis, immune gene analysis, immunogenicity analysis and gene sets enrichment analysis (GSEA). This comprehensive analysis of biomarkers for immunotherapy can be carried out by a click of CAMOIP, and the software should greatly encourage the further development of immunotherapy. CAMOIP fills the gap between the big data of cancer genomics based on immunotherapy and providing comprehensive information to users, helping to release the value of current ICI-treated data resources. CAMOIP can be found in https://www.camoip.net.


title: AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

abstract: Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a dominant phasing method for the crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database for finding suitable templates for phasing. However, such sequence information is not always available such as when proteins are crystallized with unknown binding partner proteins or when the crystal is that of a contaminant. The recent development of AlphaFold has resulted in the availability of predicted protein structures for all proteins from twenty species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough for sequence-independent phasing of diffraction data from two crystallization contaminants for which we had not identified the protein. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained which allowed the identification and structure determination of both structures, YncE and YadF. Our results advocate a general utility of AlphaFold-predicted structure database with respect to crystallographic phasing.

category: biochemistry


title: A case for glycerol as an acceptable additive to cryoEM samples

abstract: Buffer composition and sample preparation guidelines for cryo-electron microscopy are geared toward maximizing imaging contrast and reducing electron beam-induced motion. These pursuits often involve the minimization or complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, we show that inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by a [~]2.3 [A] reconstruction of mouse apoferritin ([~]500 kDa) and a [~]3.3 [A] reconstruction of rabbit muscle aldolase ([~]160 kDa) in presence of 20% v/v glycerol. While we found that generating thin ice that is amenable for high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion nor an inability to pick particles. Overall, our findings indicate glycerol should not be discounted as a cryoEM sample buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.

category: biophysics


title: Universal inverse square relationship between heart rate variability and heart rate

abstract: In our previous study, we analyzed heart rate variability and heart rate from a large variety of cardiac preparations (including humans, living animals, Langendorff-perfused isolated hearts, and single sinoatrial nodal cells) in diverse species, combining our data with those of previously published articles. The analysis revealed that regardless of conditions, heart rate variability (for the purposes of the study assessed as standard deviation of beat-to-beat intervals) vs. heart rate follows a universal exponential decay-like relationship. Numerical simulations of diastolic interval variability by adding a randomly fluctuating term (Iper) to net current revealed a similar relationship. In the present study, using a Taylor series, we found that this relationship is, in fact, inverse square, and we derive an explicit formula for the standard deviation (sd) of the cycle length (CL) as a function of heart rate (HR) with biophysically meaningful parameters: sd(CL)=sd(Iper)*(60,000/mean(HR) -APD)^2/({Delta}V*C), where CL is in ms, HR in beats per minute, Iper in pA, APD in ms is an average AP duration of pacemaker cells, C in pF is cell membrane capacitance, and {Delta}V is the magnitude of diastolic depolarization in mV. This relationship gives direct insight into heart rate variability mechanisms at the basic level of individual pacemaker cells, i.e. their intrinsic CL variability linked to stochastic operation of ion channels (both Ca release and cell membrane channels) generating Iper. Our explicit formula may be also used for a more precise biomedical interpretation of heart rate variability after respective corrections for heart rate.


title: Deep Learning identifies new morphological patterns of Homologous Recombination Deficiency in luminal breast cancers from whole slide images.

abstract: Homologous Recombination DNA-repair deficiency (HRD) is a well-recognized marker of platinum-salt and PARP inhibitor chemotherapies in ovarian and breast cancers (BC). Causing high genomic instability, HRD is currently determined by BRCA1/2 sequencing or by genomic signatures, but its morphological manifestation is not well understood. Deep Learning (DL) is a powerful machine learning technique that has been recently shown to be capable of predicting genomic signatures from stained tissue slides. However, DL is known to be sensitive to dataset biases and lacks interpretability. Here, we present and evaluate a strategy to control for biases in retrospective cohorts. We train a deep-learning model to predict the HRD in a controlled cohort with unprecedented accuracy (AUC: 0.86) and we develop a new visualization technique that allows for automatic extraction of new morphological features related to HRD. We analyze in detail the extracted morphological patterns that open new hypotheses on the phenotypic impact of HRD.


title: Multi-omic Characterization of Pancreatic Cancer-Associated Macrophage Polarization Reveals Deregulated Metabolic Programs Driven by the GMCSF-PI3K Pathway

abstract: The pancreatic ductal adenocarcinoma (PDA) microenvironment is composed of a variety of cell types and marked by extensive fibrosis and inflammation. Tumor-associated macrophages (TAM) are abundant, and they are important mediators of disease progression and invasion. TAMs are polarized in situ to a tumor promoting and immunosuppressive phenotype via cytokine signaling and metabolic crosstalk from malignant epithelial cells and other components of the tumor microenvironment (TME). However, the specific distinguishing features and functions of TAMs remain poorly defined. Here, we generated tumor-educated macrophages (TEM) in vitro and performed detailed, multi-omic characterization (i.e. transcriptomics, proteomics, metabolomics). Our results reveal unique genetic and metabolic signatures of TEMs, the veracity of which were queried against our in-house single cell RNA sequencing (scRNA-seq) dataset of human pancreatic tumors. This analysis identified expression of novel, metabolic TEM markers in human pancreatic TAMs, including ARG1, ACLY, and TXNIP. We then utilized our TEM model system to study the role of mutant Kras signaling in cancer cells on TEM polarization. This revealed an important role for GM-CSF and lactate on TEM polarization, molecules released from cancer cells in a mutant Kras-dependent manner. Lastly, we demonstrate that GM-CSF dysregulates TEM gene expression and metabolism through PI3K-AKT pathway signaling. Collectively, our results define new markers and programs to classify pancreatic TAMs, how these are engaged by cancer cells, and the precise signaling pathways mediating polarization.


title: The influence of age and sex on the pre-treatment immune microenvironment of a carcinogen induced murine model of bladder cancer

abstract: The incidence of urothelial carcinoma of the bladder is four times higher in males than females; however, females tend to present with a more aggressive disease, a poorer response to immunotherapy and suffer worse clinical outcomes. Recent findings have demonstrated sexual dimorphism in the tumor immune microenvironment of non-muscle invasive bladder cancer and associated clinical outcomes. However, a significant gap in knowledge remains with respect to the current pre-clinical modeling approaches and more precisely recapitulating these differences towards improved therapeutic design. Based on the similarities in mucosal immune physiology between humans and mice, we evaluated the sex and age-related immune alterations in healthy murine bladders. Bulk-RNA sequencing and multiplex immunofluorescence based spatial immune profiling of healthy murine bladders from male and female mice of age groups spanning young to old showed a highly altered immune landscape that exhibited sex and age associated differences, particularly in the context of B cell associated responses. Spatial profiling using markers specific to macrophages, T lymphocytes, B lymphocytes, activated dendritic cells, high endothelial venules, myeloid cells and the PD-L1 immune checkpoint showed sex and age associated differences. Bladders from healthy older female mice showed a higher number of tertiary lymphoid structures (TLSs) compared to both young female and male equivalents. Spatial immune profiling of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen exposed male and female bladders from young and old mice revealed a similar frequency of TLS formation, sex differences in the bladder immune microenvironment and, age differences in latency of tumor induction. These findings support the incorporation of sex and age as factors in pre-clinical modeling of NMIBC and will potentially advance the field of immunotherapeutic drug development to improve clinical outcomes in NMIBC.


title: OrBITS: A High-throughput, time-lapse, and label-free drug screening platform for patient-derived 3D organoids

abstract: Patient-derived organoids are invaluable for fundamental and translational cancer research and holds great promise for personalized medicine. However, the shortage of available analysis methods, which are often single-time point, severely impede the potential and routine use of organoids for basic research, clinical practise, and pharmaceutical and industrial applications. Here, we report the development of a high-throughput automated organoid analysis platform that allows for kinetic monitoring of organoids, named Organoid Brightfield Identification-based Therapy Screening (OrBITS). The combination of computer vision with a convolutional network machine learning approach allowed for the detection and tracking of organoids in routine extracellular matrix domes, advanced Gri3D(R)-96 well plates, and high-throughput 384-well microplates, solely based on brightfield imaging. We used OrBITS to screen chemotherapeutics and targeted therapies, and incorporation of a fluorescent cell death marker, revealed further insight into the mechanistic action of the drug, a feature not achievable with the current gold standard ATP-assay. This manuscript describes the validation of the OrBITS deep learning analysis approach against current standard assays for kinetic imaging and automated analysis of organoids. OrBITS, as a scalable, high-throughput technology, would facilitate the use of patient-derived organoids for drug development, therapy screening, and guided clinical decisions for personalized medicine. The developed platform also provides a launching point for further brightfield-based assay development to be used for fundamental research.


title: Engineered bacteria detect tumor DNA in vivo

abstract: Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to diseases such as gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can easily access the entire gastrointestinal tract via oral administration8, and they can produce outputs that can be noninvasively measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangement11, has been used to allow bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells and organoids. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered orally or rectally into mice injected with orthotopic donor CRC organoids. We observe horizontal gene transfer from the tumor to the sensor bacteria in vivo, allowing their detection in stool. The sensor bacteria achieved 100% discrimination between mice with and without CRC using both delivery methods. Our findings establish a framework for biosensing applications that require the detection of mutations or organisms within environments that are difficult to sample. In addition, the platform can be readily expanded to include in situ production and delivery of therapeutic payloads at the detection site.

category: synthetic biology


title: Engineered bacteria detect tumor DNA


title: Kif11 overexpression rescues cognition, long-term potentiation, and spine defects in mouse and cell models of Alzheimer's disease

abstract:
Competitive inhibition of kinesin motor proteins by amyloid-beta (A{beta}) may contribute to alterations in the neuronal microtubule cytoskeleton that can disrupt plasticity mechanisms required for learning and memory, such as long-term potentiation (LTP), thus contributing to synaptic dysfunction and cognitive impairments associated with Alzheimers disease (AD). Here, we tested the hypothesis that overexpression of the microtubule motor protein KIF11 (Kinesin-5/Eg5) will rescue A{beta}-mediated synaptic dysfunction and cognitive impairments. We found that overexpression of Kif11 prevented spatial learning and LTP deficits in the 5xFAD mouse model of AD and rescued A{beta}-mediated decreases in postsynaptic dendritic spine density in neuronal cultures. Together, these data suggest that KIF11 function is important for preserving synaptic structures and functions that are critical for learning and memory and for protection against A{beta}-mediated loss of cognition in AD.

HighlightsO_LIDeficits in cognition and long-term potentiation in the 5xFAD mouse model of Alzheimers disease are prevented by Kif11 overexpression.
C_LIO_LIA{beta}-mediated dendritic spine loss is blocked by Kif11 overexpression.
C_LI


title: Nanodomain-mediated lateral sorting drives polarization of the small GTPase ROP2 in the plasma membrane of root hair cells

abstract: Formation of root hairs involves the targeted recruitment of the cellular growth machinery to the root hair initiation domain (RHID), a specialized site at the plasma membrane (PM) of trichoblast cells. Early determinants in RHID establishment are small GTPases of the Rho-of-plants (ROP) protein family, which are required for polarization of downstream effectors, membrane modification and targeted secretion during tip growth. It remains, however, not fully understood how ROP GTPases themselves are polarized. To investigate the mechanism underlying ROP2 recruitment, we employed Variable Angle Epifluorescence Microscopy (VAEM) and exploited mCitrine fluorophore blinking for single molecule localization, particle tracking and super-resolved imaging of the trichoblast plasma membrane. We observed the association of mCit-ROP2 within distinct membrane nanodomains, whose polar occurrence at the RHID was dependent on the presence of the RopGEF GEF3, and found a gradual, localized decrease of mCit-ROP2 protein mobility that preceded polarization. We provide evidence for a step-wise model of ROP2 polarization that involves (i) an initial non-polar recruitment to the plasma membrane via interactions with anionic phospholipids, (ii) ROP2 assembly into membrane nanodomains independent of nucleotide-binding state and, sub-sequently, (iii) lateral sorting into the RHID, driven by GEF3-mediated localized reduction of ROP2 mobility.


title: Essential functions of mosquito ecdysone importers in development and reproduction

abstract: The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, 3, and 4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.


title: A transcriptome atlas of the mouse iris at single cell resolution defines cell types and the genomic response to pupil dilation

abstract: The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmological diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting, constricted, and dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell-types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.


title: Genetic-epigenetic interactions in paternal transgenerational inheritance of metabolic disorders

abstract:
ObjectiveParental environmental exposures can strongly influence descendant risks for adult disease. How paternal obesity changes the sperm chromatin leading to the acquisition of metabolic disease in offspring remains controversial and ill-defined. The objective of this study was to assess: (1) whether obesity induced by a high-fat diet alters sperm histone methylation; (2) whether paternal obesity can induce metabolic disturbances across generations; (3) whether there could be cumulative damage to the sperm epigenome leading to enhanced metabolic dysfunction in descendants; and (4) whether obesity-sensitive regions associate with embryonic epigenetic and transcriptomic profiles. Using a genetic mouse model of epigenetic inheritance, we investigated the role of histone H3 lysine 4 methylation (H3K4me3) in the paternal transmission of metabolic dysfunction. This transgenic mouse overexpresses the histone demethylase enzyme KDM1A in the developing germline and has an altered sperm epigenome at the level of histone H3K4 methylation. We hypothesized that challenging transgenic sires with a high-fat diet would further erode the sperm epigenome and lead to enhanced metabolic disturbances in the next generations.

MethodsTo assess whether paternal obesity can have inter- or transgenerational impacts, and if so, to identify potential mechanisms of this non-genetic inheritance, we used wildtype C57BL/6NCrl and transgenic males with a pre-existing altered sperm epigenome. To induce obesity, sires were fed either a control or high-fat diet (10% or 60% kcal fat, respectively) for 10-12 weeks, then bred to wildtype C57BL/6NCrl female fed a regular diet. F1 and F2 descendants were characterized for metabolic phenotypes by examining the effects of paternal obesity by sex, on body weight, fat mass distribution, the liver transcriptome, intraperitoneal glucose and insulin tolerance tests. To determine whether obesity altered the F0 sperm chromatin, native chromatin immunoprecipitation-sequencing targeting H3K4me3 was performed. To gain insight into mechanisms of paternal transmission, we compared our sperm H3K4me3 profiles with embryonic and placental chromatin states, histone modification and gene expression profiles.

ResultsObesity-induced alterations in H3K4me3 occurred at genes implicated in metabolic, inflammatory, and developmental processes. These processes were associated with offspring metabolic dysfunction and corresponded to genes enriched for H3K4me3 in embryos, and overlapped embryonic and placenta gene expression profiles. Transgenerational susceptibility to metabolic disease was only observed when obese F0 had a pre-existing modified sperm epigenome. This coincided with increased H3K4me3 alterations in sperm and more severe phenotypes affecting their offspring.

ConclusionsOur data suggest sperm H3K4me3 might serve as a metabolic sensor that connects paternal diet with offspring phenotypes via the placenta. This non-DNA based knowledge of inheritance has the potential to improve our understanding of how environment shapes heritability and may lead to novel routes for the prevention of disease. This study highlights the need to further study the connection between the sperm epigenome, placental development and childrens health.


title: Sperm Histone H3 Lysine 4 tri-methylation serves as a metabolic sensor of paternal obesity and is associated with the inheritance of metabolic dysfunction


title: HOIL-1-catalysed ubiquitylation of unbranched glucosaccharides and its activation by ubiquitin oligomers

abstract: HOIL-1, a component of the Linear Ubiquitin Assembly Complex (LUBAC), ubiquitylates serine and threonine residues in proteins, forming ester bonds (Kelsall et al, 2019, PNAS 116, 13293-13298). Here we report that mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, cardiac muscle and other organs, indicating that HOIL-1s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL-1 monoubiquitylates glycogen and 1:4-linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester-linked ubiquitylation. The HOIL-1-catalysed monoubiquitylation of maltoheptaose was accelerated >100-fold by Met1-linked or Lys63-linked ubiquitin oligomers, which interact with the catalytic RBR domain of HOIL-1. HOIL-1 also transferred preformed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL-1, bound to amylose resin in vitro, suggesting a potential function in targeting HOIL-1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal by glycophagy to prevent precipitation as polyglucosan.


title: Predicting Transmembrane (TM) Domain Dimer Structures using Martini 3

abstract: Determination of the structure and dynamics of transmembrane (TM) domains of single-transmembrane receptors is key to understanding their mechanism of signal transduction across the plasma membrane. Although many studies have been performed on isolated soluble extra- and intracellular receptor domains in aqueous solutions, limited knowledge exists on the lipid embedded TM region. In this study, we predict the assembly of alternate configurations of receptor TM domain dimers using the Martini 3 force field for coarse-grain (CG) molecular dynamic simulations. This recent version of Martini has new bead types and sizes, which allows more accurate predictions of molecular packing and interactions compared to the previous version. Our results with Martini 3 simulations show overall good agreement with ab initio predictions using PREDDIMER and with available NMR derived structures for TM helix dimers. Understanding and predicting the association TM domains may help us to better understand the signalling mechanism of TM receptors, in turn providing the opportunity for development of new pharmaceuticals, some of which are peptide based.


title: The histone deacetylase activity of HDAC1/2 is required to safeguard zygotic genome activation in mice and cattle

abstract: The genome is transcriptionally inert at fertilization and must be activated through a remarkable developmental process called zygotic genome activation (ZGA). The gene expression pattern formed over the course of ZGA is required for establishing totipotency in early embryos and subsequent development. Substantial epigenetic reprogramming contributes significantly to the pronounced change in gene expression during ZGA, however the mechanism has yet to be resolved. Here, we find histone deacetylase 1 and 2 (HDAC1/2) are critical histone modifiers that regulate ZGA through the histone deacetylase activity. Notably, we show that H3K27ac level declines dramatically during ZGA with a dynamic change in its genome-wide distribution. In mouse embryos, ectopic expression of HDAC1/2 dominant negative mutant leads to a failure of H3K27ac removal and a developmental arrest at 2-cell stage. RNA-seq results reveal a remarkable transcriptomic change with 6565 differentially expressed genes identified. Further analysis shows 64% of down-regulated genes are ZGA genes and 49% of up-regulated genes are developmental genes. Low input ChIP-seq analysis exhibits an increase and decrease of H3K27ac enrichment at the promoter region of up- and down-regulated genes, respectively. Moreover, HDAC1 mutants prohibited removal of broad H3K4me3 domain via impeding the expression of Kdm5s during ZGA. Importantly, the developmental block can be greatly overcome through injection of Kdm5b mRNA and expression of the majority of dysregulated genes partially corrected. Similar functional significance of HDAC1/2 in ZGA is conserved in bovine embryos. Together, we propose that HDAC1/2 is indispensable for mouse and bovine ZGA via creating correct transcriptional repressive and active states.


title: Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells

abstract:
Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of protein targets for peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted LC-MS-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.

ImportanceThe frontline treatments for malaria are combination therapies based on the peroxide antimalarial, artemisinin. Concerningly, artemisinin resistance has emerged in malaria-endemic regions, and now poses a major threat to malaria treatment and eradication efforts. New medicines are urgently required to replace the artemisinins, and some of the most advanced candidates are the fully synthetic peroxide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel). The mechanism of action of peroxide antimalarials involves the reductive activation of the peroxide bond by intra-parasitic haem, but there is no consensus regarding the specific protein targets of the resulting radical species for artemisinins and/or the ozonides. This study provides a comprehensive and unbiased chemoproteomic profile of over 400 target proteins, and confirms the specific impact of peroxide antimalarials on redox metabolism. The key role of redox targets is particularly relevant considering that the mechanism of artemisinin resistance appears to involve modulation of peroxide activation and redox homeostasis.


title: A modular toolset for electrogenetics

abstract: Synthetic biology research and its industrial applications rely on the deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and an electrode), allowing for the direct integration of electronics with complex biological processes for a variety of new applications. However, the use of electrogenetic systems is limited by poor activity, tunability and standardisation. Here, we have developed a variety of genetic and electrochemical tools that facilitate the design and vastly improve the performance of electrogenetic systems. We developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We then constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Finally, we demonstrated electrochemical activation of gene expression in aerobic conditions utilising a novel, modular bioelectrochemical device. This toolset provides researchers with all the elements needed to design and build optimised electrogenetic systems for specific applications.


title: Germinal center activity and B cell maturation promote protective antibody responses against Plasmodium pre-erythrocytic infection

abstract: Blocking Plasmodium, the causative agent of malaria, at the asymptomatic pre-erythrocytic stage would abrogate disease pathology and prevent transmission. Rodent-infectious species of Plasmodium such as P. yoelii (Py) serve as key tools to study vaccine efficacy and disease biology in immune-competent experimental animals. Here we evaluated the differences in vaccine-elicited humoral immunity in two widely used, and vastly diverged, inbred mouse strains, BALB/cJ and C57BL/6J, and identified immunological factors associated with protection. We vaccinated with Py circumsporozoite protein (CSP), the major surface antigen on the sporozoite, and evaluated protective efficacy after mosquito bite challenge. Vaccination achieved 60% sterile protection and otherwise delayed blood stage patency in BALB/cJ mice, whereas; all C57BL/6J mice were infected similar to controls. Interestingly, protection was mediated by antibodies, and could be passively transferred from immunized BALB/cJ mice into naive C57BL/6J. Dissection of the underlying immunological features of protection revealed early deficits in antibody titers and polyclonal avidity in C57BL/6J mice. Additionally, PyCSP-vaccination in BALB/cJ induced a significantly higher proportion of antigen-specific B-cells and class-switched memory B-cell (MBCs) populations than in C57BL/6J mice. Strikingly, C57BL/6J mice also had markedly fewer germinal center experienced, CSP-specific class-switched MBCs compared to BALB/cJ mice. Analysis of the IgG {gamma} chain repertoires by next generation sequencing in PyCSP-specific memory B-cell repertoires also revealed higher somatic hypermutation rates in BALB/cJ mice than in C57BL/6J mice. These findings indicate that BALB/cJ mice achieved higher levels of B cell maturation in response to vaccination with PyCSP, which likely enabled the development of protective antibody responses. Overall, our study indicates that germinal center activity and B cell maturation are key processes in the development of vaccine-elicited protective antibodies against CSP.


title: Single cell multi-omics reveals early elevated function and multiple fates within human progenitor exhausted CD8+ T cells

abstract:
T-cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of subjects with primary infection using single cell multi-omics to identify the functions and phenotypes of HCV-specific CD8+ T cells. Early elevated IFN-{gamma} response against the transmitted virus was associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome we discovered progenitors of early-exhaustion with intermediate expression of PD-1. Intra clonal analysis revealed distinct trajectories with multiple fates suggesting evolutionary plasticity of precursor cells. These findings challenge current paradigm on the contribution of CD8+ T cells to HCV disease outcome and provide data for future studies on T-cell differentiation in human infections.

One sentence summaryProgenitors of T-cell exhaustion in acute HCV infection


title: Overriding defective FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in diabetic wound

abstract: Infection is a major co-morbidity that contributes to impaired healing in diabetic wounds. Although impairments in diabetic neutrophils have been blamed for this co-morbidity, what causes these impairments and whether they can be overcome, remain largely unclear. Diabetic neutrophils, extracted from diabetic individuals, exhibit chemotaxis impairment but this peculiar functional impairment has been largely ignored because it appears to contradict the clinical findings which blame excessive neutrophil influx (neutrophilia) as a major impediment to healing in chronic diabetic ulcers. Here, we report that exposure to glucose in diabetic range results in impaired chemotaxis signaling through the FPR1 chemokine receptor in neutrophils, culminating in reduced chemotaxis and delayed neutrophil trafficking in wound in diabetic animals, and rendering diabetic wound vulnerable to infection. We further show that at least some auxiliary chemokine receptors remain functional under diabetic conditions and their engagement by the pro-inflammatory cytokine CCL3, overrides the requirement for FPR1 signaling and substantially improves infection control by jumpstarting the neutrophil response toward infection, and stimulates healing in diabetic wound. We posit that CCL3 may have real therapeutic potential for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds.


title: Transplantation of bacteriophages from ulcerative colitis patients shifts the gut bacteriome and exacerbates severity of DSS-colitis

abstract: Inflammatory bowel diseases (IBDs) including Crohns disease (CD) and ulcerative colitis (UC) are characterized by chronic and debilitating gut inflammation. Altered bacterial communities of the intestine are strongly associated with IBD initiation and progression. The gut virome, which is primarily composed of bacterial viruses (bacteriophages, phages) is thought to be an important factor regulating and shaping microbial communities in the gut. While alterations in the gut virome have been observed in IBD patients, the contribution of these viruses to alterations in the bacterial community and heightened inflammatory responses associated with IBD patients remains largely unknown. Here, we performed in vivo microbial cross-infection experiments to follow the effects of fecal virus-like particles (VLPs) isolated from UC patients and healthy controls on bacterial diversity and severity of experimental colitis in human microbiota-associated (HMA) mice. Shotgun metagenomics confirmed that several phages were transferred to HMA mice, resulting in treatment-specific alterations in the gut virome. VLPs from healthy and UC patients also shifted gut bacterial diversity of these mice, an effect that was amplified during experimental colitis. VLPs isolated from UC patients specifically altered the relative abundance of several bacterial taxa previously implicated in IBD progression. Additionally, UC VLP administration heightened colitis severity in HMA mice, as indicated by shortened colon length and increased pro-inflammatory cytokine production. Importantly, this effect was dependent on intact VLPs. Our findings build on recent literature indicating that phages are dynamic regulators of bacterial communities in the gut and implicate the intestinal virome in modulating intestinal inflammation and disease.

category: microbiology


title: A well conserved archaeal B-family polymerase functions as a mismatch and lesion extender

abstract: B-family DNA polymerases (PolBs) of different groups are widespread in Archaea and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that are poorly characterized is PolB2 whose members occur in many archaea but are predicted as an inactivated form of DNA polymerase. Herein, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase harbors a robust nucleotide incorporation activity, but devoid of the 3-5 exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. Together, these data suggested Dpo2 functions as a mismatch and lesion extender, representing a novel type of PolB that is primarily involved in DNA damage repair in Archaea. Insights were also gained into the functional adaptation of the motif C in the mismatch extension of the B-family DNA polymerases.


title: New auranofin analogs with antibacterial properties against Burkholderia clinical isolates

abstract: Bacteria of the genus Burkholderia include pathogenic Burkholderia mallei, Burkholderia pseudomallei and the Burkholderia cepacia complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species B. cenocepacia is associated with one of the worst clinical outcomes. Following the repurposing of auranofin as an antibacterial against Gram-positive bacteria, we previously synthetized auranofin analogs with activity against Gram-negatives. In this work, we show that two auranofin analogs, MS-40S and MS-40, have antibiotic activity against Burkholderia clinical isolates. The compounds are bactericidal against B. cenocepacia and kill stationary-phase cells and persisters without selecting for multistep resistance. Caenorhabditis elegans and Galleria mellonella tolerated high concentrations of MS-40S and MS-40, demonstrating that these compounds have low toxicity in these model organisms. In summary, we show that MS-40 and MS-40S have the potential to be effective therapeutic options to treat Burkholderia infections.


title: Novel prokaryotic sensing and regulatory system employing previously unknown nucleic acids-based receptors

abstract:
The present study describes a previously unknown universal signaling and regulatory system, which we named TRB receptor system. This system is responsible for sensing, remembering, and regulating cell responses to various chemical, physical or biological stimuli. It controls cell survival, variability, reproduction, adaptation, genome changes, and gene transfer. Importantly, the TRB-receptor system is responsible for the formation and maintenance of cell memory, as well the ability to "forget" preceding events. The system is composed of DNA- and RNA-based receptors located outside the membrane named "TezRs", as well as reverse transcriptases and integrases. The sensory and regulatory functions of TezRs enable the TRB-receptor system to control all major aspects of bacterial behavior, such as growth, biofilm formation and dispersal, utilization of nutrients including xenobiotics, virulence, chemo- and magnetoreception, response to external factors (e.g., temperature, UV, light and gas content), mutation events, phage-host interaction and recombination activity. Additionally, it supervises the function of other receptor-mediated signaling pathways. Transcriptome analysis revealed that the loss of different TezRs instigates significant alterations in gene expression.

HIGHLIGHTSThe TRB-receptor system regulates bacterial sensing and response to various stimuli.

The TRB-receptor system is responsible for maintenance and loss of cell memory.

The TRB-receptor system comprises DNA- and RNA-based "TezRs" receptors.

The TRB-receptor system relies on reverse transcriptases and recombinases.

The TRB-receptor system oversees other receptor-mediated signaling pathways.

TezRs are implicated in cell mutation and recombination events.


title: Even allocation of benefits stabilizes microbial community engaged in metabolic division of labor

abstract: Metabolic division of labor (MDOL) is commonly observed in microbial communities, where a metabolic pathway is sequentially implemented by several members similar to an assembly line. Uncovering the assembly rules of the community engaged in MDOL is crucial for understanding its ecological contribution, as well as for engineering high-performance microbial communities. To investigate the assembly of the community engaged in MDOL, we combined mathematical modelling with experimentations using synthetic microbial consortia. We built a theoretical framework predict the assembly of MDOL system, and derived a simple rule: to maintain co-existence of the MDOL members, the populations responsible for former steps should hold a growth advantage (m) over the  private benefit (n) of the population responsible for last step, and the steady-state frequency of the last population is determined by the quotient of n and m. Our experiments further indicated that our theoretical framework accurately predicted the stability and assembly of our engineered synthetic consortia that degrade naphthalene through two-step or multi-step MDOL. Our results demonstrate that the assembly of microbial community engaged in MDOL is determined by a limited number of parameters. This quantitative understanding provides novel insights on designing and managing stable microbial systems to address grand challenges facing human society in agriculture, degradation of the environment, and human health.


title: Metagenomic identification of viral sequences in laboratory reagents

abstract:
2.Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterize the complete  infectome (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing ("metatranscriptomics") we documented the presence of contaminant viral sequences in multiple libraries of  blank negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank libraries, as well as contaminating sequences from the RNA virus families Totiviridae, Tombusviridae and Lentiviridae. These data suggest that the contamination of common laboratory reagents is likely widespread and can comprise a wide variety of viruses.

3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

1.5 RepositoriesThe viral genome sequence data generated in this study has been deposited in the NCBI database under accession numbers MZ824225-MZ824237. Sequence reads are available at the public Sequence Read Archive (SRA) database with accession SRX6803604 and under the BioProject accession PRJNA735051 reference numbers SRR14737466-71 and BioSample numbers SAMN20355437-40.

category: molecular biology


title: Exosomal MFGE8 from high glucose induced endothelial cells is involved in calcification/senescence of vascular smooth muscle cells

abstract: Vascular calcification/aging is a crucial feature of diabetic macro vasculopathy, resulting in serious cardiovascular diseases. The calcification/senescence of vascular smooth muscle cells (VSMCs) induced by hyperglycemia can cause diabetic vascular calcification/aging. However, the mechanism of VSMCs calcification/senescence involved in diabetic vascular calcification/aging remains unknown. The purpose of this study was to determine how the high glucose (HG) information in circulating blood is transmitted from vascular endothelial cells (ECs) to VSMCs, which are not contacted with blood directly. Exosomes have attracted much attention for their vital roles in regulating cell-to-cell communication. In this study, we found that milk fat globule epidermal growth factor 8 (MFGE8) was enriched in high glucose induced human umbilical vein endothelial cell exosomes (HG-HUVEC-Exo) and regulate VSMCs calcification/senescence, characterized by up-regulated expressions of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2), as well as the increased mineralized nodules and senescence-associated {beta}-galactosidase (SA-{beta}-gal) positive cells. Upstream mechanism studies showed that sirtuin1 (SIRT1) was involved in VSMCs calcification/senescence by affecting the expression of MFGE8. We also found that inflammatory response mediated by IL-1{beta}, IL-6, and IL-8 was closely associated with MFGE8 and played a key role in regulating HG-HUVEC-Exo-induced VSMCs calcification/senescence. These findings provide a new insight into the mechanism of exosomal MFGE8 as a potential preventive and therapeutic target for the intervention of diabetic vascular calcification/aging.


title: Convergent patterns of gene expression and protein evolution associated with adaptation to desert environments in rodents

abstract: Desert specialization has arisen multiple times across rodents and is often associated with a suite of convergent phenotypes, including modification of the kidneys to mitigate water loss. However, the extent to which phenotypic convergence in desert rodents is mirrored at the molecular level is unknown. Here, we sequenced kidney mRNA and assembled transcriptomes for three pairs of rodent species to search for convergence in gene expression and amino acid sequence associated with adaptation to deserts. We conducted phylogenetically-independent comparisons between a desert specialist and a non-desert relative in three families representing [~]70 million years of evolution. Overall, patterns of gene expression faithfully recapitulated the phylogeny of these six taxa. However, we found that 8.6% of all genes showed convergent patterns of expression evolution between desert and non-desert taxa, a proportion that is much higher than expected by chance. In addition to these convergent changes, we observed many species-pair specific changes in gene expression indicating that different instances of adaptation to deserts include a combination of unique and shared changes. Patterns of protein evolution revealed a small number of genes showing evidence of positive selection, the majority of which did not show convergent changes in gene expression. Overall, our results suggest convergent changes in gene regulation play a primary role in the complex trait of desert adaptation in rodents.


title: Newly developed MAGIC population allows identification of strong associations and candidate genes for anthocyanin pigmentation in eggplant

abstract: MAGIC populations facilitate the genetic dissection of complex quantitative traits in plants and are valuable breeding materials. We report the development of the first eggplant MAGIC population (S3MEGGIC; 8-way), constituted by 420 S3 individuals developed from the intercrossing of seven cultivated eggplant (Solanum melongena) and one wild relative (S. incanum) parents. The S3MEGGIC recombinant population was genotyped with the eggplant 5k probes SPET platform and phenotyped for anthocyanins presence in vegetative plant tissues (PA) and fruit epidermis (FA), and for the light-sensitive anthocyanic pigmentation under the calyx (PUC). The 7,724 filtered high-confidence SNPs confirmed a low residual heterozygosity (6.87%) and a lack of genetic structure in the S3MEGGIC population, including no differentiation among subpopulations carrying cultivated or wild cytoplasm. Inference of haplotype blocks of the nuclear genome revealed an unbalanced representation of founder genomes, suggesting cryptic selection in favour or against specific parental genomes. GWAS analysis for PA, FA and PUC detected strong associations with two MYB genes similar to MYB113 involved in the anthocyanin biosynthesis pathway and with a COP1 gene, which encodes for a photo-regulatory protein and may be responsible for the PUC phenotype. Evidence was found of a duplication of an ancestral MYB113 gene with a translocation from chromosome 10 to chromosome 1. Parental genotypes for the three genes were in agreement with the candidate genes identification performed in the S3MEGGIC population. Our new eggplant MAGIC population is the largest recombinant population in eggplant and is a powerful tool for eggplant genetics and breeding studies.

category: genetics


title: Evaluating the power and limitations of genome-wide association mapping in C. elegans

abstract: A central goal of evolutionary genetics in Caenorhabditis elegans is to understand the genetic basis of traits that contribute to adaptation and fitness. Genome-wide association (GWA) mappings scan the genome for individual genetic variants that are significantly correlated with phenotypic variation in a population, or quantitative trait loci (QTL). GWA mappings are a popular choice for quantitative genetic analyses because the QTL that are discovered segregate in natural populations. Despite numerous successful mapping experiments, the empirical performance of GWA mappings has not, to date, been formally evaluated for this species. We developed an open-source GWA mapping pipeline called NemaScan and used a simulation-based approach to provide benchmarks of mapping performance among wild C. elegans strains. Simulated trait heritability and complexity determined the spectrum of QTL detected by GWA mappings. Power to detect smaller-effect QTL increased with the number of strains sampled from the C. elegans Natural Diversity Resource (CeNDR). Population structure was a major driver of variation in GWA mapping performance, with populations shaped by recent selection exhibiting significantly lower false discovery rates than populations composed of more divergent strains. We also recapitulated previous GWA mappings of experimentally validated quantitative trait variants. Our simulation-based evaluation of GWA performance provides the community with critical context for pursuing quantitative genetic studies using CeNDR to elucidate the genetic basis of complex traits in C. elegans natural populations.


title: De-novo chromosome level assembly of plant genomes from long read sequence data

abstract:
Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. The chromosome level assembly of the contigs generated from long read sequencing has involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (PacBio HiFi) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii. a genome that has been used as model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig and the other 6 assembled into 2-4 main contigs. The small number of chromosome breaks appear to be due to highly repetitive regions of ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now seems possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.

Significance statement (of up to two sentences)De novo assembly of near complete chromosome level plant genomes is now possible using current long read sequencing and assembly tools.


title: Evolutionary innovations through gain and loss of genes in the ectomycorrhizal Boletales

abstract:
O_LIIn this study, we aim to identify genomic traits of the transitions to the ectomycorrhizal ecology within the Boletales, one of the most diverse lineages of symbiotrophic fungi.
C_LIO_LIWe sequenced the genomes and compared the gene repertoires of symbiotrophic Boletales species to their saprotrophic brown-rot relatives. We also reconstructed gene duplication/loss histories along a time-calibrated phylogeny.
C_LIO_LIWe showed that the rate of gene duplication is constant along the backbone of Boletales phylogeny with large loss events in lineages leading to several families. The rate of gene family expansion sharply increased in the late Miocene and mostly took place in Boletaceae.
C_LIO_LIMost of the ectomycorrhizal Boletales are characterized by a large genome size due to transposable element (TE) expansions and a reduction in the diversity of plant cell wall degrading enzymes (PCWDEs) compared to their brown-rot relatives. However, several species in the Boletaceae, Paxillaceae and Boletinellaceae have kept a substantial set of endoglucanases and LPMOs acting on cellulose/hemicellulose and fungal polysaccharides suggesting that they may partly decompose organic matter by a combined activity of oxidative and hydrolytic enzymes.
C_LIO_LIThe present study provides novel insights on our understanding of the mechanisms that influence the evolutionary diversification of boletes and symbiosis evolution.
C_LI


title: Chromosome-scale genome assembly of Eustoma grandiflorum, the first complete genome sequence in family Gentianaceae

abstract: Eustoma grandiflorum (Raf.) Shinn., is an annual herbaceous plant native to the southern United States, Mexico, and the Greater Antilles. It has a large flower with a variety of colors and an important flower crop. In this study, we established a chromosome-scale de novo assembly of E. grandiflorum by integrating four genomic and genetic approaches: (1) Pacific Biosciences (PacBio) Sequel deep sequencing, (2) error correction of the assembly by Illumina short reads, (3) scaffolding by chromatin conformation capture sequencing (Hi-C), and (4) genetic linkage maps derived from an F2 mapping population. The 36 pseudomolecules and unplaced 64 scaffolds were created with total length of 1,324.8 Mb. Full-length transcript sequencing was obtained by PacBio Iso-Seq sequencing for gene prediction on the assembled genome, Egra_v1. A total of 36,619 genes were predicted on the genome as high confidence HC) genes. Of the 36,619, 25,936 were annotated functions by ZenAnnotation. Genetic diversity analysis was also performed for nine commercial E. grandiflorum varieties bred in Japan, and 254,205 variants were identified. This is the first report of the construction of reference genome sequences in E. grandiflorum as well as in the family Gentianaceae.


title: Comparative Genome Analysis of 'Candidatus Phytoplasma luffae' Reveals the Influential Roles of Potential Mobile Units in Phytoplasma Evolution

abstract: Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of  Candidatus Phytoplasma luffae strain NCHU2019 that is associated with witches broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has an exceptionally repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ~25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for neofunctionalization or subfunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.


title: BINANA 2.0: Characterizing Protein/Ligand Interactions in Python and JavaScript

abstract: BINding ANAlyzer (BINANA) is an algorithm for identifying and characterizing protein/ligand interactions and other factors that contribute to binding. We recently updated BINANA to make the algorithm more accessible to a broader audience. We have also ported the Python3 codebase to JavaScript, thus enabling BINANA analysis in the web browser. As proof of principle, we created a web-browser application so students and chemical-biology researchers can quickly visualize receptor/ligand complexes and their unique binding interactions.


title: Cyclic mechanical stresses alter erythrocyte membrane composition and microstructure and trigger macrophage phagocytosis

abstract: Red blood cells (RBCs) are cleared from the circulation when they become damaged or display aging signals targeted by macrophages. This process occurs mainly in the spleen, where blood flows through submicrometric constrictions called inter-endothelial slits (IES), subjecting RBCs to large-amplitude deformations. In this work, we circulated RBCs through microfluidic devices containing microchannels that replicate the IES. The cyclic mechanical stresses experienced by the cells affected their biophysical properties and molecular composition, accelerating cell aging. Specifically, RBCs quickly transitioned to a more spherical, less deformable phenotype that hindered microchannel passage, causing hemolysis. This transition was associated with the release of membrane vesicles, which self-extinguished as the spacing between membrane-cytoskeleton linkers became tighter. Proteomics analysis of the mechanically aged RBCs revealed significant losses of essential proteins involved in antioxidant protection, gas transport, and cell metabolism. Finally, we show that these changes made mechanically aged RBCs more susceptible to macrophage phagocytosis. These results provide a comprehensive model to explain how physical stress induces RBC clearance in the spleen.

category: bioengineering


title: Transcriptional networks underpinning ploidy related increased leaf potassium in neo-tetraploids

abstract: Neo-tetraploid Arabidopsis thaliana have elevated leaf potassium (K) driven by processes within the root. The root transcriptome of neo-tetraploids is distinct from diploids, with evidence of altered K homeostasis. Mutational analysis revealed that the canonical K-uptake transporters AKT1 and HAK5 are not required for this elevated leaf K in neo-tetraploids, while the endodermis, root hairs, and SOS signaling are. Contrasting the root transcriptomes of neo-tetraploids and diploids of mutants that block the neo-tetraploid K phenotype, allowed us to identify 91 differentially expressed genes associated with elevated leaf K in neo-tetraploids. This set of genes connects WGD to elevated leaf K, and is enriched in functions such as cell wall and Casparian strip development, and ion-transport, in the endodermis, root hairs, and procambium. This gene set provides tools to test the intriguing idea of recreating the physiological effects of WGD within a diploid genome.

category: plant biology


title: Effects of vestibular stimulation on gait stability and variability

abstract: Vestibular information modulates muscle activity during gait, presumably to contribute to stability. If this is the case, stronger effects of perturbing vestibular stimulation on gait stability can be expected for narrow-base walking (which needs more control) than for normal walking and smaller effects for wide-base walking (which needs less control). An important mechanism to stabilize gait in the mediolateral direction is to coordinate foot placement based on a sensory estimate of the center of mass (CoM) state, to which vestibular information appears to contribute. We, therefore, expected that stochastic vestibular stimulation would decrease the correlation between foot placement and CoM state. In fourteen healthy participants, we measured the kinematics of the trunk (as a proxy of CoM), and feet, while they walked on a treadmill in six conditions: control, narrow-base, and wide-base, each with and without stochastic electrical vestibular stimulation (EVS) (peak amplitude of 5 mA; RMS of ~ 1.2 mA; frequency band of 0 to 25 Hz). Stochastic EVS decreased local dynamic stability. Foot placement correlated stronger with trunk motion during walking with EVS in the control and narrow-base conditions. However, residual variance in foot placement was increased with EVS indicating less accurate foot placement, although this effect was not significantly different between step width conditions. Thus, vestibular information contributes to control of gait stability, but this contribution is not modulated by step width.


title: Effects of vestibular stimulation on control of gait stability


title: ON/OFF domains shape receptive fields in mouse visual cortex

abstract: In higher mammals, thalamic afferents to primary visual cortex (area V1) segregate according to their responses to increases (ON) or decreases (OFF) in luminance1-4. This organization induces columnar, ON/OFF domains postulated to provide a scaffold for the emergence of orientation tuning2,5-15. To further test this idea, we asked whether ON/OFF domains exist in mouse V1 - a species containing orientation tuned, simple cells, like those found in other mammals16-19. Here we show that mouse V1 is indeed parceled into ON/OFF domains. Revealingly, fluctuations in the relative density ON/OFF neurons on the cortical surface mirror fluctuations in the relative density of ON/OFF receptive field centers on the visual field. In each cortical volume examined, the average of simple-cell receptive fields correlates with the difference between the average of ON and OFF receptive fields7. Moreover, the local diversity of simple-cell receptive fields is explained by a model in which neurons linearly combine a small number of ON and OFF signals available in their cortical neighborhoods15,20. Altogether, these findings indicate that ON/OFF domains originate in fluctuations of the spatial density of ON/OFF inputs on the visual field which, in turn, shapes the structure of receptive fields10-13,21-23.


title: Lateralized phenotypic differences in females in the intrahippocampal kainic acid mouse model of temporal lobe epilepsy

abstract:
Clinical evidence indicates that patients with temporal lobe epilepsy (TLE) often show differential outcomes of comorbid conditions in relation to the lateralization of the seizure focus. A particularly strong relationship exists between the side of seizure focus and the propensity for distinct reproductive endocrine comorbidities in women with TLE. Therefore, here we evaluated whether targeting of left or right dorsal hippocampus for intrahippocampal kainic acid (IHKA) injection, a model of TLE, produces different outcomes in hippocampal granule cell dispersion, body weight gain, and multiple measures of reproductive endocrine dysfunction in female mice. One, two, and four months after IHKA or saline injection, in vivo measurements of estrous cycles and weight were followed by ex vivo examination of hippocampal dentate granule cell dispersion, circulating ovarian hormone and corticosterone levels, ovarian morphology, and pituitary gene expression. IHKA mice with right-targeted injection (IHKA-R) showed greater granule cell dispersion and pituitary Fshb expression compared to mice with left-targeted injection (IHKA-L). By contrast, pituitary expression of Lhb and Gnrhr were higher in IHKA-L mice compared to IHKA-R, but these values were not different from respective saline-injected controls. IHKA-L mice also showed an increased rate of weight gain compared to IHKA-R mice. Increases in estrous cycle length, however, were similar in both IHKA-L and IHKA-R mice. These findings indicate that although major reproductive endocrine dysfunction phenotypes present similarly after targeting left or right dorsal hippocampus for IHKA injection, distinct underlying mechanisms based on lateralization of epileptogenic insult may contribute to produce similar emergent reproductive endocrine outcomes.

HighlightsO_LIGreater granule cell dispersion with right-sided IHKA injection
C_LIO_LIChanges in pituitary gene expression vary with side of injection
C_LIO_LIIncreased weight gain after left-sided injection
C_LIO_LISimilar estrous cycle disruption after injection of left or right hippocampus
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title: Lateralized phenotypic differences after intrahippocampal kainic acid injection in female mice


title: A linear neural circuit for light avoidance in Drosophila larvae

abstract: Understanding how neural circuits underlie behaviour is challenging even in the era of the connectome because it requires a combined approach encompassing anatomical and functional analyses. This is exemplified in studying the circuit underlying the light-avoidance behaviour displayed by the larvae of the fruit fly Drosophila melanogaster. While this behaviour is robust and the nervous system relatively simple, only bits and pieces of the circuit have been delineated1. Indeed, some studies resulted in contradicting conclusions regarding the contributions of various neuronal types to this behaviour2,3. Here we devise trans-Tango MkII, a new version of the transsynaptic circuit tracing and manipulation tool trans-Tango4. We implement trans-Tango MkII in anatomical tracing and combine it with circuit epistasis analysis. We use neuronal inhibition to test necessity of particular neuronal types for light-avoidance. We complement these experiments by selective neuronal activation to examine sufficiency in rescuing light-avoidance deficiencies exhibited by photoreceptor mutants. Together, our studies reveal a four-order, linear circuit for light-avoidance behaviour connecting the light-detecting photoreceptors with a pair of neuroendocrine cells via two types of clock neurons. Our combined approach could be readily expanded to other larval circuits. Further, this strategy provides the framework for studying more complex nervous systems and behaviours.


title: Local neuronal excitation and global inhibition during epileptic fast ripples in humans.

abstract: Understanding the neuronal basis of epileptiform activity is a major challenge in neurology. Interictal epileptiform discharges are associated with fast ripples (FRs, >200 Hz) in the local field potential (LFP) and are a promising marker of the epileptogenic zone. Here, by using a novel hybrid macro-micro depth electrode, combining classic depth recording of LFP and two or three tetrodes enabling up to 15 neurons in local circuits to be recorded simultaneously, we have characterized neuronal responses to FRs on the same hybrid and other electrodes targeting other brain regions. While FRs were associated with increased neuronal activity in local circuits only, they were followed by inhibition in large-scale networks. Neuronal responses to FRs were homogeneous in local networks but differed across brain areas. Similarly, post-FR inhibition varied across recording locations and subjects and was shorter than typical inter-FR intervals, suggesting that this inhibition is a fundamental refractory process for the networks. These findings demonstrate that FRs engage local and global networks and point to network features that pave the way for new diagnostic and therapeutic strategies.


title: A curved manifold orients rotational dynamics in motor cortex

abstract: Controlling arm movements requires complex, time-varying patterns of muscle activity 1,2. Accordingly, the responses of neurons in motor cortex are complex, time-varying, and heterogeneous during reaching 2-4. When examined at the population level, patterns of neural activity evolve over time according to dynamical rules 5,6. During reaching, these rules have been argued to be "rotational" 7 or variants thereof 8,9, containing coordinated oscillations in the spike rates of individual neurons. While these models capture key aspects of the neural responses, they fail to capture others - accounting for only 20-50% of the neural response variance. Here, we consider a broader class of dynamical models. We find that motor cortex dynamics take an unexpected form: there were 3-4 rotations at fixed frequencies in M1 and PMd explaining more than 90% of neural responses, but these rotations occurred in different portions of state space when movements differ. These rotations appear to reflect a curved manifold of fixed points in state space, around which dynamics are locally rotational. These fixed-frequency rotations obeyed a simple relationship with movement: the orientation of rotations in motor cortex activity were related almost linearly to the movement the animal made, allowing linear decoding of reach kinematic time-courses on single trials. This model constitutes a fundamentally novel way to consider pattern generation: like placing a record player in a large bowl, the frequency of activity is fixed, but the location of motor cortex activity on a curved manifold sets the orientation of locally-rotational dynamics. This system simplifies motor control, helps reconcile conflicting frameworks for interpreting motor cortex, and enables greatly improved neural decoding.