title: Newly Emergent Apelin Expressing Endothelial Stem-like Cells Orchestrate Lung Microvascular Repair

abstract:
QuestionWe sought to define the mechanism underlying lung microvascular regeneration in a severe acute lung injury (ALI) model induced by selective lung endothelial cell ablation.

MethodsChanges in lung cell populations and gene expression profiles were determined in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs using single-cell RNA sequencing at baseline (day 0) and days 3, 5 and 7 after lung EC ablation.

ResultsEight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline, and general capillary (gCap) ECs expressing the apelin receptor. Intratracheal instillation of DT resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. At 3 days post injury, a novel gCap population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned to proliferative ECs, expressing apelin receptor together with the pro-proliferative transcription factor, FoxM1. This progenitor-like cell population was responsible for the rapid replenishment of all depleted EC populations by 7 days post injury, including aerocytes which play a critical role in re-establishment of the air-blood barrier. Treatment with an apelin receptor antagonist prevented recovery and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair.

ConclusionThe lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by signaling between newly emergent apelin-expressing gCap endothelial stem-like cells and highly proliferative, apelin receptor positive endothelial progenitors.

Take-Home messageUsing sublethal lung endothelial cell (EC) ablation, we show for the first that EC regeneration and resolution of acute lung injury is orchestrated by novel apelin-expressing, gCap endothelial stem-like cells by a mechanism requiring apelin signaling.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

A schematic representation of EC populations contributing to microvascular repair. At baseline (Day 0), there are two main alveolar groups of capillary ECs: larger apelin positive aCap ECs, termed aerocytes, that play a key structural role in forming the air-blood barrier; and smaller apelin receptor (Aplnr) expressing gCap ECs, which are found in the thicker regions at the corners of the alveoli. After DT-induced EC ablation, there is a marked depletion of both EC populations and the appearance of novel transitional and transient populations. At Day 3, there is the appearance of stem-like gCap ECs that paradoxically express apelin, but not its receptor, and are characterized by various stem and progenitor cell markers but show no evidence of proliferation. By Day 5, these transition to ECs expressing Aplnr which have a strong proliferative phenotype, as evidenced by FoxM1 and Ki67 expression, and then rapidly replenish depleted EC pools, including aCap ECs, by Day 7. This transition is orchestrated by the interaction of apelin with its receptor as a critical mechanism in lung microvascular regeneration after EC injury. AT1 = alveolar type -1 epithelial cell; AT2 = alveolar type-2 epithelial cell; APLNR = apelin receptor; ANGPT2 = angiopoietin 2; EPCR = Endothelial protein C receptor.


title: Novel Apelin-expressing gCap Endothelial Stem-like Cells Orchestrate Lung Microvascular Repair

category: cell biology


title: miR-9a regulates levels of both rhomboid mRNA and protein in the early Drosophila melanogaster embryo

abstract: MicroRNAs have subtle and combinatorial effects on the expression levels of their targets. Studying the consequences of a single microRNA knockout often proves difficult as many such knockouts exhibit phenotypes only under stress conditions. This has led to the hypothesis that microRNAs frequently act as buffers of noise in gene expression. Observing and understanding buffering effects requires quantitative analysis of microRNA and target expression in single cells. To this end, we have employed single molecule fluorescence in situ hybridization, immunofluorescence, and high-resolution confocal microscopy to investigate the effects of miR-9a loss on the expression of the serine-protease rhomboid in Drosophila melanogaster early embryos. Our single-cell quantitative approach shows that rhomboid mRNA exhibits the same spatial expression pattern in WT and miR-9a knockout embryos, although the number of mRNA molecules per cell is higher when miR-9a is absent. However, the level of rhomboid protein shows a much more dramatic increase in the miR-9a knockout. Specifically, we see accumulation of rhomboid protein in miR-9a mutants by stage 5, much earlier than in WT. The data therefore show that miR-9a functions in the regulation of rhomboid activity by both inducing mRNA degradation and inhibiting translation in the blastoderm embryo. Temporal regulation of neural proliferation and differentiation in vertebrates by miR-9 is well-established. We suggest that miR-9 family microRNAs are conserved regulators of timing in neurogenic processes. This work shows the power of single-cell quantification as an experimental tool to study phenotypic consequences of microRNA mis-regulation.

category: developmental biology


title: Therapeutic base and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa

abstract: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). We used base and prime editing to correct mutations in fibroblasts from two patients (Pat1, who carried a c.3631C>T mutation in one allele, and Pat2, who carried a c.2005C>T mutation in one allele). We applied adenine base editors (ABEs) to correct the pathogenic mutation or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that both ABE- and PE-mediated correction of COL7A1 mutations restored full-length C7 expression, reversed the impaired adhesion and proliferation exhibited by the patient-derived fibroblasts, and, following transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice, led to C7 deposition within the dermal-epidermal junction. These results suggest that base and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.

category: bioengineering


title: Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer

abstract: Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells have lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells altered expression of numerous other proteins implicated in metastatic progression. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels and decreased disease-free survival. These findings suggest that downregulation of lamin A/C may influence both biochemical and physical properties of the cell to promote breast cancer metastatic progression.

category: cancer biology


title: Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy

abstract: Anti-CTLA-4 antibodies such as ipilimumab were among the first immune-oncology agents to show significantly improved outcomes for patients. However, existing anti-CTLA-4 therapies fail to induce a response in a majority of patients and can induce severe, immune-related adverse events. It has been assumed that checkpoint inhibition, i.e., blocking the interaction between CTLA-4 and its ligands, is the primary mechanism of action for ipilimumab. In this study we present evidence that checkpoint inhibition is not a primary mechanism of action for efficacy of anti-CTLA-4 antibodies. Instead, the primary mechanism for efficacy is FcR-mediated Treg depletion in the tumor microenvironment. First, we identified a monoclonal antibody (mAb) that binds to CTLA-4 at an epitope that differs from ipilimumabs by only a few amino acids, yet has limited checkpoint inhibitor activity. Surprisingly, the weak checkpoint inhibitor has superior anti-tumor activity compared to ipilimumab in a murine model. The weak checkpoint inhibitor also induces less Treg proliferation and has increased ability to induce in vitro FcR signaling and in vivo depletion of intratumoral Tregs. Further experiments showed that the enhanced FcR activity of the weak checkpoint inhibitor likely contributes to its enhanced anti-tumor activity. Importantly, we also showed that weak checkpoint inhibition was associated with lower toxicity in murine models. Our work suggests that new anti-CTLA-4 drugs should be optimized for Treg depletion rather than checkpoint inhibition.

category: immunology


title: Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

abstract: Human pluripotent stem cell derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for derivation of skeletal muscle cells and their utility in three-dimensional skeletal muscle organoid formation as well as skeletal muscle tissue engineering. Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors of hypaxial origin followed by primary and secondary fetal myogenesis into hypaxial muscle with development of a satellite cell pool and evidence for innervation in vitro. Skeletal muscle organoids faithfully recapitulate all steps of embryonic myogenesis in 3D Tissue engineered muscle exhibits organotypic maturation and function, advanced by thyroid hormone. Regenerative competence was demonstrated in a cardiotoxin injury model with evidence of satellite cell activation as underlying mechanism. Collectively, we introduce a hypaxial muscle model with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease, and repair.


title: The use of high-resolution computed tomography to explore age-related trabecular change in human ribs

abstract:
High-resolution computed tomography was used to explore age-related trabecular change in male human ribs ranging in age from 20-95 years (Mean=55 years, SD=21.634 years) from the Texas State Donated Skeletal Collection (TXSTDSC). Two regions of interest (ROIs), midshaft (50%) and anterior (75%) were extracted from each scan to analyze age-related trabecular change. Dragonfly V4.1 was used to isolate cortical bone volumes of interest (VOIs) and three trabecular VOIs for each ROI; one each along the cutaneous cortex, the center of the medullary cavity, and the pleural cortex. Each trabecular VOI was analyzed for bone volume fraction (BV/TV), trabecular thickness (TbTh), trabecular spacing (TbSp), connectivity density (Conn.D), and degree of anisotropy (DA), within and between the 50 and 75% ROIs. Overall, the cutaneous VOIs at both the 50% and 75% ROIs exhibited greater BV/TV, TbTh, and Conn.D when compared to the center and pleural VOIs. All results are consistent with expected biomechanical strain on human ribs. Both trabecular variables and cortical bone volume are only weakly associated with age. These results show that 3D analysis of trabecular bone volume does not improve visualization or understanding of trabecular bone changes with age over traditional 2D methods. Future research should incorporate female samples to explore sex-related trabecular change variation.

HIGHLIGHTSO_LI3D analysis of change in trabecular structure along the rib length and with age
C_LIO_LITrabecular spacing at midshaft shows highest correlations with age-at-death
C_LIO_LIRelative cortical area is more strongly correlated with age in anterior ribs than at midshaft
C_LI


title: In vivo evidence that SORL1, encoding the endosomal recycling receptor SORLA, can function as a causal gene in Alzheimer's Disease

abstract: The few established causal genes in Alzheimers disease (AD), mutations in APP and PSENs, have been functionally characterized using biomarkers, capturing an in vivo profile reflecting the diseases initial preclinical phase. SORL1, a gene encoding the endosome recycling receptor SORLA, epidemiologically behaves as a causal gene when truncating mutations lead to partial loss of protein function. Here, in an effort to test whether SORL1 can indeed function as an AD causal gene, we used CRISPR-Cas9-based gene editing to develop a novel model of SORL1 haploinsufficiency in Gottingen Minipigs taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young minipigs was found to phenocopy the preclinical in vivo profile of AD observed with other causal genes, resulting in spinal fluid abnormalities in A{beta} and tau, with no evident neurodegeneration or amyloid plaque formation. These studies provide functional support that SORL1 is a bona fide causal gene in AD, and when taken together with recent insight on other AD-causal genes, support the idea that dysfunctional endosomal recycling is a dominant pathogenic pathway in the disease.

category: neuroscience


title: Modelling complex population structure using F-statistics and Principal Component Analysis

abstract: Human genetic diversity is shaped by our complex history. Data-driven methods such as Principal Component Analysis (PCA) are an important population genetic tool to understand this method. Here, I contrast PCA with a set of statistics motivated by trees (F-statistics). Here, I show that these two methods are closely related, and I derive explicit connections between the two approaches. I show that F-statistics have a simple geometrical interpretation in the context of PCA, and that orthogonal projections are the key concept to establish this link. I illustrate my results on two examples, one of local, and one of global human diversity. In both examples, I find that just using the first few PCs provides good population structure is sparse, and only a few components contribute to most statistics. Based on these results, I develop novel visualizations that allow for investigating specific hypotheses, checking the assumptions of more sophisticated models. My results extend F-statistics to non-discrete populations, moving towards more complete and less biased descriptions of human genetic variation.

category: evolutionary biology


title: WDR82-binding long non-coding RNA lncEry controls mouse erythroid differentiation and maturation

abstract: Hematopoietic differentiation is controlled by both genetic and epigenetic regulators. Long non-coding RNAs (lncRNAs) have been demonstrated to be important for normal hematopoiesis, but their function in erythropoiesis needs to be further explored. We profiled the transcriptomes of 16 murine hematopoietic cell populations by deep RNA-sequencing and identified a novel lncRNA, Gm15915, that was highly expressed in erythroid-related progenitors and erythrocytes. For this reason, we named it lncEry. We also identified a novel lncEry isoform, which was also the principal transcript that has not been reported before. LncEry depletion impaired erythropoiesis, indicating the important role of the lncRNA in regulating erythroid differentiation and maturation. Mechanistically, we found that lncEry interacted with WD repeat-containing protein 82 (WDR82) to promote the transcription of Klf1 and globin genes and thus control the early and late stages of erythropoiesis, respectively. These findings identified lncEry as an important player in the transcriptional regulation of erythropoiesis.


title: Determining molecular archetype composition and expression from bulk tissues with unsupervised deconvolution

abstract: Complex tissues are composite ecological systems whose components interact with each other to create a unique physiological or pathophysiological state distinct from that found in other tissue microenvironments. To explore this ground yet dynamic state, molecular profiling of bulk tissues and mathematical deconvolution can be jointly used to characterize heterogeneity as an aggregate of molecularly distinct tissue or cell subtypes. We first introduce an efficient and fully unsupervised deconvolution method, namely the Convex Analysis of Mixtures - CAM3.0, that may aid biologists to confirm existing or generate novel scientific hypotheses about complex tissues in many biomedical contexts. We then evaluate the CAM3.0 functional pipelines using both simulations and benchmark data. We also report diverse case studies on bulk tissues with unknown number, proportion and expression patterns of the molecular archetypes. Importantly, these preliminary results support the concept that expression patterns of molecular archetypes often reflect the interactive not individual contributions of many known or novel cell types, and unsupervised deconvolution would be more powerful in uncovering novel multicellular or subcellular archetypes.

category: bioinformatics


title: Pharmacokinetics of caffeine: A systematic analysis of reported data for application in metabolic phenotyping and liver function testing

abstract: Caffeine is by far the most ubiquitous psychostimulant worldwide found in tea, coffee, cocoa, energy drinks, and many other beverages and food. Caffeine is almost exclusively metabolized in the liver by the cytochrome P-450 enzyme system to the main product paraxanthine and the additional products theobromine and theophylline. Besides its stimulating properties, two important applications of caffeine are metabolic phenotyping of cytochrome P450 1A2 (CYP1A2) and liver function testing. An open challenge in this context is to identify underlying causes of the large inter-individual variability in caffeine pharmacokinetics. Data is urgently needed to understand and quantify confounding factors such as lifestyle (e.g. smoking), the effects of drug-caffeine interactions (e.g. medication metabolized via CYP1A2), and the effect of disease. Here we report the first integrative and systematic analysis of data on caffeine pharmacokinetics from 148 publications and provide a comprehensive high-quality data set on the pharmacokinetics of caffeine, caffeine metabolites, and their metabolic ratios in human adults. The data set is enriched by meta-data on the characteristics of studied patient cohorts and subjects (e.g. age, body weight, smoking status, health status), the applied interventions (e.g. dosing, substance, route of application), measured pharmacokinetic time-courses, and pharmacokinetic parameters (e.g. clearance, half-life, area under the curve). We demonstrate via multiple applications how the data set can be used to solidify existing knowledge and gain new insights relevant for metabolic phenotyping and liver function testing based on caffeine. Specifically, we analyzed (i) the alteration of caffeine pharmacokinetics with smoking and use of oral contraceptives; (ii) drug-drug interactions with caffeine as possible confounding factors of caffeine pharmacokinetics or source of adverse effects; (iii) alteration of caffeine pharmacokinetics in disease; and (iv) the applicability of caffeine as a salivary test substance by comparison of plasma and saliva data. In conclusion, our data set and analyses provide important resources which could enable more accurate caffeine-based metabolic phenotyping and liver function testing.

category: pharmacology and toxicology


title: Electrocorticography based monitoring of anaesthetic depth in mice

abstract: To improve animal welfare and data quality and reproducibility during research conducted under anaesthesia, anaesthetic depth in laboratory animals must be precisely monitored and controlled. While a variety of methods have been developed to estimate the depth of anaesthesia in humans, such tools for monitoring anaesthetic depth in laboratory animals remain limited. Here we propose an epidural electrocorticogram-based monitoring system that accurately tracks the depth of anesthesia in mice receiving inhalable isoflurane anaesthesia. Several features of the electrocorticogram signals exhibit robust modulation by the concentration of the administered anesthetic, notably, corticocortical coherence serves as an excellent indicator of anaesthetic depth. We developed a gradient boosting regressor framework that utilizes the extracted features to accurately estimate the depth of anaesthesia. Our method for feature extraction and estimation is conducted with a latency of only ten seconds, establishing a system for the real-time tracking of anaesthetic depth in mice.


title: Arousal impacts distributed hubs modulating the integration of brain functional connectivity

abstract: Even when subjects are at rest, it is thought that brain activity is organized into distinct brain states during which reproducible patterns are observable. Yet, it is unclear how to define or distinguish different brain states. A potential source of brain state variation is arousal, which may play a role in modulating functional interactions between brain regions. Here, we use simultaneous resting state functional magnetic resonance imaging and pupillometry to study the impact of arousal levels indexed by pupil area on the integration of large-scale brain networks. We employ a novel sparse dictionary learning-based method to identify hub regions participating in between-network integration stratified by arousal, by measuring k-hubness, the number (k) of functionally overlapping networks in each brain region. We show evidence of a brain-wide decrease in between-network integration and inter-subject variability at low relative to high arousal, with differences emerging across regions of the frontoparietal, default mode, motor, limbic, and cerebellum networks. State-dependent changes in k-hubness relate to the actual patterns of network integration within these hubs, suggesting a brain state transition from high to low arousal characterized by global synchronization and reduced network overlaps. We demonstrate that arousal is not limited to specific brain areas known to be directly associated with arousal regulation, but instead has a brain-wide impact that involves high-level between-network communications.


title: Single-nucleus sequencing reveals enriched expression of genetic risk factors sensitises Motor Neurons to degeneration in ALS

abstract: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterised by a progressive loss of motor function. While it is known that the eponymous spinal sclerosis observed upon autopsy is the result of Cortico-Spinal Motor Neuron (CSMN) degeneration, it remains unclear why this neuronal subtype is selectively affected. To understand the unique molecular properties that sensitise deep-layer CSMNs to ALS, we performed RNA sequencing of 79,169 single nuclei from the frontal cortex of patients and controls. In unaffected individuals, we found that expression of ALS risk genes was most significantly enriched only in THY1+ presumptive CSMNs and not in other cortical cell types. In patients, these genetic risk factors, as well as additional genes involved in protein homeostasis and stress responses, were significantly induced in THY1+ CSMNs and a wider collection of deep layer neurons, but not in neurons with more superficial identities. Examination of oligodendroglial and microglial nuclei also revealed patient-specific gene expression changes We show microglial alterations can in part be explained by interactions with degenerating neurons. Overall, our findings suggest the selective vulnerability of CSMNs is due to a "first over the line" mechanism by which their intrinsic molecular properties sensitise them to genetic and mechanistic contributors to degeneration.


title: VCP protects neurons from proteopathic seeding

abstract: Uptake and spread of proteopathic seeds, such as S, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of S seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase S seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of S seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to S since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.


title: Prolonged morphological expansion of spiny-rayed fishes following the end-Cretaceous

abstract: Spiny-rayed fishes (Acanthomorpha) dominate modern marine habitats and comprise more than a quarter of all living vertebrate species1-3. It is believed that this dominance resulted from explosive lineage and phenotypic diversification coincident with the Cretaceous-Paleogene (K-Pg) mass-extinction event4. It remains unclear, however, if living acanthomorph diversity is the result of a punctuated burst or gradual accumulation of diversity following the K-Pg. We assess these hypotheses with a time-calibrated phylogeny inferred using ultraconserved elements from a sampling of species that represent over 91% of all acanthomorph families, as well as an extensive body shape dataset of extant species. Our results indicate that several million years after the end-Cretaceous, acanthomorphs underwent a prolonged and significant expansion of morphological disparity primarily driven by changes in body elongation, and that acanthomorph lineages containing the bulk of the living species diversity originated throughout the Cenozoic. These acanthomorph lineages radiated into distinct regions of morphospace and retained their iconic phenotypes, including a large group of laterally compressed reef fishes, fast-swimming open-ocean predators, bottom-dwelling flatfishes, seahorses, and pufferfishes. The evolutionary success of spiny-rayed fishes is the culmination of a post K-Pg adaptive radiation in which rates of lineage diversification were decoupled from periods of high phenotypic disparity.


title: A complete reference genome improves analysis of human genetic variation

abstract:
Compared to its predecessors, the Telomere-to-Telomere CHM13 genome adds nearly 200 Mbp of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome to clinical and functional study. Here we demonstrate how the new reference universally improves read mapping and variant calling for 3,202 and 17 globally diverse samples sequenced with short and long reads, respectively. We identify hundreds of thousands of novel variants per sample--a new frontier for evolutionary and biomedical discovery. Simultaneously, the new reference eliminates tens of thousands of spurious variants per sample, including up to 12-fold reduction of false positives in 269 medically relevant genes. The vast improvement in variant discovery coupled with population and functional genomic resources position T2T-CHM13 to replace GRCh38 as the prevailing reference for human genetics.

One Sentence SummaryThe T2T-CHM13 reference genome universally improves the analysis of human genetic variation.

category: genomics


title: Complete genomic and epigenetic maps of human centromeres

abstract:
Existing human genome assemblies have almost entirely excluded highly repetitive sequences within and near centromeres, limiting our understanding of their sequence, evolution, and essential role in chromosome segregation. Here, we present an extensive study of newly assembled peri/centromeric sequences representing 6.2% (189.9 Mb) of the first complete, telomere-to-telomere human genome assembly (T2T-CHM13). We discovered novel patterns of peri/centromeric repeat organization, variation, and evolution at both large and small length scales. We also found that inner kinetochore proteins tend to overlap the most recently duplicated subregions within centromeres. Finally, we compared chromosome X centromeres across a diverse panel of individuals and uncovered structural, epigenetic, and sequence variation at single-base resolution across these regions. In total, this work provides an unprecedented atlas of human centromeres to guide future studies of their complex and critical functions as well as their unique evolutionary dynamics.

One-sentence summaryDeep characterization of fully assembled human centromeres reveals their architecture and fine-scale organization, variation, and evolution.


title: Development of photo-lenalidomide for cellular target identification

abstract:
The thalidomide analog lenalidomide is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photo-lenalidomide, a lenalidomide probe with a photo-affinity label and enrichment handle, for target identification by chemical proteomics. After evaluating a series of lenalidomide analogs, we identified a specific amide linkage to lenalidomide that allowed for installation of the desired functionality, while preserving the substrate degradation profile, phenotypic anti-proliferative and immunomodulatory properties of lenalidomide. Photo-lenalidomide maintains these properties by enhancing binding interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using photo-lenalidomide, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells, and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by photolenalidomide and forms a complex with CRBN in the presence of lenalidomide, but is itself not ubiquitylated or degraded. These data point to the potentially broader array of substrates induced by ligands to CRBN that may or may not be degraded, which can be revealed by the highly translatable application of photo-lenalidomide and chemical proteomics in additional biological settings.

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category: biochemistry


title: How drugs modulate the performance of the human heart

abstract: Many drugs interact with ion channels in the cells of the heart and trigger heart rhythm disorders with potentially fatal consequences. Computational modeling can provide mechanistic insight into the onset and propagation of drug-induced arrhythmias, but the effect of drugs on the mechanical behavior of the heart remains poorly understood. Here we establish a multiphysics framework that integrates the biochemical, electrical, and mechanical effects of drugs from single cardiac cells to the overall response of the whole heart. For the example of the drug dofetilide, we show that drug concentrations of 3.0x and 4.8x increase the heart rate to 122 and 114 beats per minute, increase the myofiber stretches up to 10%, and decrease tissue relaxation by 6%. Strikingly, the drug-induced interventricular and atrial-ventricular dyssynchrony results in a 2.5% decreased and 7% increased cardiac output, respectively. Our results demonstrate the potential for multiphysics, multiscale modeling towards understanding the mechanical implications of drug-induced arrhythmias. Knowing how differing drug concentrations affect the performance of the heart has important clinical implications in drug safety evaluation and personalized medicine.

category: biophysics


title: Long chain lipids facilitate insertion of large nanoparticles into membranes of small unilamellar vesicles

abstract: Insertion of hydrophobic nanoparticles into phospholipid bilayers is limited to small particles that can incorporate into the hydrophobic membrane core in between the two lipid leaflets. Incorporation of nanoparticles above this size limit requires development of challenging surface engineering methodologies. In principle, increasing membrane thickness should facilitate incorporation of larger nanoparticles. Here we explore the effect of very long phospholipids (C24:1) in small unilamellar vesicles, on the membrane insertion efficiency of nanoparticles in the range of 5-13 nm in diameter. To this end, we improved an existing vesicle preparation protocol and utilized cryogenic electron microscopy imaging to examine the mode of interaction and to evaluate the membrane insertion efficiency of membrane-inserted nanoparticles.


title: Inulin nanostructures: The sweet-spot of carbohydrate self-assembly

abstract: Supramolecular architecture formation by the self-assembly of proteins and nucleic acids is well studied. Yet, the spontaneous organization of oligosaccharides, the most common polymers in nature, is less explored. Here, using inulin as a model, we identify the "sweet spot" length for oligosaccharide assembly. Inulin forms discrete spheres in a concentration-dependent manner. Size-based fractionation displayed markedly different aggregation morphologies. Based on these findings, we believe that carbohydrates could become an important source for novel self-assembling materials.


title: An atlas of the Norway spruce needle seasonal transcriptome

abstract:
Boreal conifers possess a tremendous ability to survive and remain evergreen during harsh winter conditions and resume growth during summer. This is enabled by coordinated regulation of major cellular functions at the level of gene expression, metabolism, and physiology. Here we present a comprehensive characterization of the annual changes in the global transcriptome of Norway spruce needles as a resource to understand needle development and acclimation processes throughout the year. In young, growing needles (May 15 - June 30), cell walls, organelles etc. were formed, and this developmental program heavily influenced the transcriptome, explained by over represented Gene Ontology (GO) categories. Later changes in gene expression were smaller but four phases were recognized: summer (July-August), autumn (September-October), winter (November-February) and spring (March-April), where over represented GO categories demonstrated how the needles acclimated to the various seasons. Changes in the seasonal global transcriptome profile were accompanied by differential expression of members of the major transcription factor families. We present a tentative model of how cellular activities are regulated over the year in needles of Norway spruce, which demonstrates the value of mining this dataset, accessible in ConGenIE together with advanced visualization tools.

Significance statementThe development of Norway spruce needles and their annual cycle of biochemical activities (photosynthesis in the summer, adaptation and survival of the harsh boreal winter) is not well understood. We use deep RNA sequencing to profile the transcriptome over the season, and show how the dataset could be used to give information about "what needles do" over the year, and are here making the dataset available for the scientific community.

Graphical abstract

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category: plant biology


title: GH18 family glycoside hydrolase Chitinase A of Salmonella facilitates bacterial invasion and survival by modulating host immune responses

abstract: Salmonella is a facultative intracellular pathogen that has co-evolved with its host and has also developed various strategies to evade the host immune responses. Salmonella recruits an array of virulence factors to escape from host defense mechanisms. Previously chitinase A (chiA) was found to be upregulated in intracellular Salmonella. Although studies show that chitinases and chitin binding proteins (CBP) of many human pathogens have a profound role in various aspects of pathogenesis, like adhesion, virulence and immune evasion, the role of chitinase in strict intravacuolar pathogen Salmonella has not yet been elucidated. In this study, we deciphered the role of chitinase of Salmonella in the pathogenesis of the serovars, Typhimurium and Typhi. Our data propose that ChiA mediated modification of the glycosylation on the epithelial cell surface facilitates the invasion of the pathogen into the epithelial cells. Further we found that ChiA aids in reactive nitrogen species (RNS) and reactive oxygen species (ROS) production in phagocytes, leading to MHCII downregulation followed by suppression of antigen presentation and antibacterial responses. In continuation of the study in animal model C. elegans, Salmonella Typhi ChiA was found to facilitate attachment to the intestinal epithelium, gut colonization and persistence by downregulating antimicrobial peptides.

category: microbiology


title: Structural insight into Marburg virus nucleoprotein-RNA complex formation

abstract: The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 [A]. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.


title: In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography

abstract: The ability to produce membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of [~] 90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified MEs and MVs in 13 species and classified several major ultrastructures: 1) tubes with a uniform diameter (with or without an internal scaffold), 2) tubes with irregular diameter, 3) tubes with a vesicular dilation at their tip, 4) pearling tubes, 5) connected chains of vesicles (with or without neck-like connectors), 6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

category: molecular biology


title: TNF-α levels in respiratory samples are associated with SARS-CoV-2 infection.

abstract:
PurposeThe aim of this study was to measure levels of IL-6 and TNF- in respiratory samples from individuals with symptoms compatible with COVID-19 and analyze their association with SARS-CoV-2 presence.

MethodsSARS-CoV-2 detection was performed using the CDC (USA) real-time RT-PCR primers, probes and protocols. Cytokine concentrations were measured using commercial reagents based on enzyme linked immunosorbent assay (ELISA).

ResultsTNF- median levels were greater in COVID19 (+) symptomatic group (5.88 (1.36 - 172.1) pg/ml) compared to COVID19 (-) symptomatic individuals (2.87 (1.45 - 69.9) pg/ml) (p=0.0003). No significant differences were shown in IL-6 median values between COVID-19 (+) and (-) symptomatic patients (5.40 (1.7 - 467) pg/ml and 6.07 (1.57 - 466.6) pg/ml respectively). In addition, increased TNF- levels (greater than 10 pg/ml), but not IL-6, were associated with SARS-CoV-2 presence (OR= 5.7; p=0.006; 95% CI= 1,551 to 19,11).

ConclusionsWe found a statistically significant association between the production of local TNF- and the presence of the virus in early stages of infection. IL-6 showed high levels in swabs from some symptomatic patients but independent from SARS-CoV-2 presence and viral load, individuals age and gender. On the contrary, TNF- evaluation confirmed the presence of inflammatory response but mostly related to COVID-19. More studies are required in order to characterize the cytokine profile expressed at the site of infection of SARS-CoV-2 and its implications in disease outcomes.


title: Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine

abstract:
The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions.

Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in immunized mice, resulting in the production of neutralizing antibodies. The immunity induced by the vaccine is sufficient to protect K18-hACE2 transgenic mice from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with RBM peptides derived from a different genetic variant of SARS-CoV-2, inducing a similarly potent neutralization activity in vaccinated mice. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.


title: Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

abstract: Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with the viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can bind comparatively stabilized to the viral RdRp active site, as it primarily forms base stacking with the template Uracil nucleotide (at +1), which is under freely fluctuations and supports a low free energy barrier of the RDV-TP insertion ([~] 1.5 kcal/mol). In comparison, the barrier ([~] 2.6 kcal/mol), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be particularly stabilized by motif B-N691, S682, and motif F-K500 with the sugar, base, and the template backbone, respectively. Although the RDV-TP insertion can be hindered by motif-F R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif-A D623, respectively.


title: Development Of The Inactivated QazCovid-In Vaccine: Protective Efficacy Of The Vaccine In Syrian Hamsters

abstract: In March 2020, the first cases of human coronavirus infection COVID-19 were registered in Kazakhstan. We isolated the SARS-CoV-2 virus from the clinical material from the patients. Subsequently, a whole virion inactivated candidate vaccine, QazCovid-in, was developed based on this virus. To obtain the vaccine, a virus grown in Vero cell culture was used, which was inactivated with formaldehyde, purified, concentrated, sterilized by filtration, and then sorbed on aluminum hydroxide gel particles. The formula virus and adjuvant in buffer saline solution was used as a vaccine. The safety and protective effectiveness of the developed vaccine was studied on Syrian hamsters. The results of the studies showed the absolute safety of the candidate vaccine on the Syrian hamsters. When studying the protective effectiveness, the developed vaccine with an immunizing dose of 5 mcg/dose of a specific antigen protected animals from wild virus at a dose of 104.5 TCID50/ml. The candidate vaccine formed virus-neutralizing antibodies in vaccinated hamsters in titers from 3.3 {+/-} 1.45 log2 to 7.25 {+/-} 0.78 log2, which were retained for 6 months (observation period) in the indicated titers. The candidate vaccine suppressed the replication of the wild virus in the body of vaccinated hamsters, protected against the development of acute pneumonia and ensured 100% survival of the animals. At the same time, no replicative virus was isolated from the lungs of vaccinated animals. At the same time, a virulent virus was isolated from the lungs of unvaccinated animals in relatively high titers, reaching 4.5 {+/-} 0.7 lg TCID50/ml. After challenge infection, 100% of unvaccinated hamsters became ill with clinical signs (stress state, passivity, tousled coat, decreased body temperature and body weight, and the development of acute pneumonia), of which 25 {+/-} 5% were fatal. The findings paved the way for testing the candidate vaccine in humans in clinical trials.


title: A drug candidate for treating adverse reactions caused by pathogenic antibodies inducible by COVID-19 virus and vaccines

abstract: In a recent study, we reported that certain anti-spike antibodies of COVID-19 and SARS-CoV viruses can have a pathogenic effect through binding to sick lung epithelium cells and misleading immune responses to attack self-cells. We termed this new pathogenic mechanism "Antibody Dependent Auto-Attack" (ADAA). This study explores a drug candidate for prevention and treatment of such ADAA-based diseases. The drug candidate is a formulation comprising N-acetylneuraminic acid methyl ester (NANA-Me), an analog of N-acetylneuraminic acid. NANA-Me acts through a unique mechanism of action (MOA) which is repairment of the missing sialic acid on sick lung epithelium cells. This MOA can block the antibodies binding to sick cells, which are vulnerable to pathogenic antibodies. Our in vivo data showed that the formulation significantly reduced the sickness and deaths caused by pathogenic anti-spike antibodies. Therefore, the formulation has the potential to prevent and treat the serious conditions caused by pathogenic antibodies during a COVID-19 infection. In addition, the formulation has potential to prevent and treat the adverse reactions of COVID-19 vaccines because the vaccines can induce similar antibodies, including pathogenic antibodies. The formulation will be helpful in increasing the safety of the vaccines without reducing the vaccines efficacy. Compared to existing antiviral drugs, the formulation has a unique MOA of targeting receptors, broad spectrum of indications, excellent safety profile, resistance to mutations, and can be easily produced.


title: Analysis of amino acid change dynamics reveals SARS-CoV-2 variant emergence

abstract: Since its emergence in late 2019, the diffusion of SARS-CoV-2 is associated with the evolution of its viral genome1,2. The co-occurrence of specific amino acid changes, collectively named  virus variant, requires scrutiny (as variants may hugely impact the agents transmission, pathogenesis, or antigenicity); variant evolution is studied using phylogenetics3-6. Yet, never has this problem been tackled by digging into data with ad hoc analysis techniques. Here we show that the emergence of variants can in fact be traced through data-driven methods, further capitalizing on the value of large collections of SARS-CoV-2 sequences. For all countries with sufficient data, we compute weekly counts of amino acid changes, unveil time-varying clusters of changes with similar - rapidly growing - dynamics, and then follow their evolution. Our method succeeds in timely associating clusters to variants of interest/concern, provided their change composition is well characterized. This allows us to detect variants emergence, rise, peak, and eventual decline under competitive pressure of another variant. Our early warning system, exclusively relying on deposited sequences, shows the power of big data in this context, and concurs to calling for the wide spreading of public SARS-CoV-2 genome sequencing for improved surveillance and control of the COVID-19 pandemic.


title: Multiplex base editing to convert TAG into TAA codons in the human genome

abstract: Large-scale recoding has been shown to enable novel amino acids, biocontainment and viral resistance in bacteria only so far. Here we extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~ 40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and multiplex editing in mammalian cells.

category: synthetic biology


title: Bidirectionally connected cores in a mouse connectome: Towards extracting the brain subnetworks essential for consciousness

abstract: Where in the brain consciousness resides remains unclear. It has been suggested that the subnetworks supporting consciousness should be bidirectionally (recurrently) connected because both feed-forward and feedback processing are necessary for conscious experience. Accordingly, evaluating which subnetworks are bidirectionally connected and the strength of these connections would likely aid the identification of regions essential to consciousness. Here, we propose a method for hierarchically decomposing a network into cores with different strengths of bidirectional connection, as a means of revealing the structure of the complex brain network. We applied the method to a whole-brain mouse connectome. We found that cores with strong bidirectional connections consisted of regions presumably essential to consciousness (e.g., the isocortical and thalamic regions, and claustrum) and did not include regions presumably irrelevant to consciousness (e.g., cerebellum). Contrarily, we could not find such correspondence between cores and consciousness when we applied other simple methods which ignored bidirectionality. These findings suggest that our method provides a novel insight into the relation between bidirectional brain network structures and consciousness.


title: Detecting the state of drowsiness induced by propofol by spatial filter and machine learning algorithm on EEG recording

abstract: To accurately measure the depth of anesthesia has been a challenge for both anesthesiologists and engineers who work on developing tools of measurements. This study aims to use a machine-learning algorithm to predict the drowsy state, a transitional depth of sedation during propofol anesthesia. The data used in this study were scalp EEG (electroencephalogram) recordings selected from the University of Cambridge Repository. Raw EEG recordings were preprocessed into power spectrum matrices one second per sample. A total of 170 samples (110 awake samples and 60 drowsy samples) were used. A CNN (Convolutional Neural Network) for the MNIST (Modified National Institute of Standards and Technology) dataset was applied on these EEG power spectrum matrices. Due to the small dataset volume, Leave-One-Out cross-validation was used to train the data. Results of the training accuracy reached 99.69%. And test accuracy averaged 96.47%. Overall, the model is able to predict the state of drowsiness during propofol anesthesia. This provides the potential to develop EEG monitoring devices with closed-loop feedback of such a machine learning algorithm that controls the titration of the dosage of anesthetic administration and the depth of anesthesia.


title: A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host

abstract: PLEs are phage parasites integrated into the chromosome of epidemic Vibrio cholerae. In response to infection by its viral host ICP1, PLE excises, replicates and hijacks ICP1 structural components for transduction. Through an unknown mechanism PLE prevents ICP1 from transitioning to rolling circle replication (RCR), a prerequisite for efficient packaging of the viral genome. Here, we characterize a PLE-encoded nuclease, NixI, that blocks phage development likely by nicking ICP1s genome as it transitions to RCR. NixI-dependent cleavage sites appear in ICP1s genome during infection of PLE(+) V. cholerae. Purified NixI demonstrates in vitro specificity for sites in ICP1s genome and NixI activity is enhanced by a putative specificity determinant co-expressed with NixI during phage infection. Importantly, NixI is sufficient to limit ICP1 genome replication and eliminate progeny production. We identify distant NixI homologs in an expanded family of putative phage satellites in Vibrios that lack nucleotide homology to PLEs but nonetheless share genomic synteny with PLEs. More generally, our results reveal a previously unknown mechanism deployed by phage parasites to limit packaging of their viral hosts genome and highlight the prominent role of nuclease effectors as weapons in the arms race between antagonizing genomes.


title: Predictions of biodiversity are improved by integrating trait-based competition with abiotic filtering

abstract: All organisms must simultaneously tolerate the environment and access limiting resources if they are to persist. Otherwise they go extinct. Approaches to understanding environmental tolerance and resource competition have generally been developed independently. Consequently, integrating the factors that determine abiotic tolerance with those that affect competitive interactions to model species abundances and community structure remains an unresolved challenge. This is likely the reason why current models of community assembly do not accurately predict species abundances and dynamics. Here, we introduce a new synthetic framework that models both abiotic tolerance and biotic competition by using functional traits, which are phenotypic attributes that influence organism fitness. First, our framework estimates species carrying capacities that vary along abiotic gradients based on whether the phenotype tolerates the local environment. Second, it estimates pairwise competitive interactions as a function of multidimensional trait differences between species and determines which trait combinations produce the most competitive phenotypes. We demonstrate that our combined approach more than doubles the explained variance of species covers in a wetland community compared to the model of abiotic tolerances alone. Trait-based integration of competitive interactions and abiotic filtering improves our ability to predict species abundances across space, bringing us closer to more accurate predictions of biodiversity structure in a changing world.

category: ecology


title: Genetic, bioacoustic and morphological analyses reveal cryptic speciation in the warbling vireo complex (Vireo gilvus: Vireonidae: Passeriformes)

abstract: Cryptic species are closely related taxa that are difficult to separate morphologically, but are reproductively isolated. Here we examine the warbling vireo complex (Vireo gilvus), a widespread songbird speculated to be comprised of more than one cryptic species. We included three taxa within the complex: two of the western (Vireo gilvus swainsonii and V. g. brewsteri) subspecies and the single eastern (V. g. gilvus) subspecies. We used mtDNA and microsatellite loci to assess the congruence of genetic data to the current subspecies boundaries. We then incorporated bioacoustic, morphometric, and ecological niche modeling analyses to further examine differences. We found two genetic groups with mtDNA analysis. Microsatellite analyses revealed four genetic groups: an eastern group, a Black Hills group and two western groups that do not agree with current western subspecies boundaries based on phenotypic data. Our results suggest that eastern and western warbling vireos have been reproductively isolated for a long period of time and therefore, may be best treated as separate species; however, more research into areas of contact to examine the presence of hybridization is advised before making a taxonomic revision. Differences between the two western genetic groups appear less clear, requiring additional research.


title: Protozoan predation drives adaptive divergence in Pseudomonas fluorescens SBW25; ecology meets experimental evolution.

abstract: Protozoan predators can affect the structure of bacterial communities, but investigations of how predation might influence bacterial evolution and antagonistic behaviours are scarce. Here, we performed a 20-day predator-prey evolution experiment on solid media to investigate the effect of continuous protozoan predation on bacterial traits using Pseudomonas fluorescens SBW25 as prey and Naegleria gruberi as an amoeboid predator. We observed the divergence of colony morphotypes coincident with an increase in bacterial grazing resistance and relative prey fitness in selected bacterial isolates. When subjected to these resistant prey, N. gruberi show reduced activity (increased encystment) and limited replication. An investigation of the mutations responsible for predation resistance reveals mutations in wspF and amrZ genes, affecting biofilm formation and motility. The bacterial mutants in the wspF gene successfully colonise the air-liquid interface and produce robust cellulose biofilms that prevent predation. The mutation in the amrZ mutant withstands predation but this variant produces low levels of cellulose and limited swarming motility. Our findings suggest that protozoan predation can profoundly influence the course of genetic and phenotypic evolution in a short period.


title: Core bone diameter in an organic implant-less technique affecting the biomechanical properties of the anterior cruciate ligament fixation; an in-vitro study

abstract:
BackgroundBone and site hold tendon inside (BASHTI) is an implant-less technique that can solve some of the problems associated with other anterior cruciate ligament (ACL) reconstructive methods. This study aims to investigate the effect of core bone diameter variation on the biomechanical properties of a reconstructed ACL using BASHTI technique.

MethodsA number of 15 laboratory samples of reconstructed ACL were built using bovine digital tendons and Sawbones blocks. Samples were divided into three groups with different core bone diameters of 8 mm, 8.5 mm, and 9 mm. The double-stranded tendon size and bone tunnel diameter were 8 mm and 10 mm, respectively. A loading scenario consisting of two cyclic loadings followed by a single cycle pull-out loading was applied to the samples simulating the after-surgery loading conditions to observe the fixation strength.

ResultsResults showed that the core bone diameter had a significant effect on the failure mode of the samples (P = 0.006) and their fixation strength (P < 0.001). Also, it was observed that the engaging length and the average cyclic stiffness (ACS) of them were influenced by the core bone diameter significantly (engaging length: P = 0.001, ACS: P = 0.007), but its effect on the average pull-out stiffness was not significant (P = 0.053).

ConclusionsIt was concluded that core bone diameter variation has a significant impact on the mechanical properties of ACL reconstruction when BASHTI technique is used, and it should be noted for surgeons who use BASHTI technique.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/452098v1_ufig1.gif" ALT="Figure 1">
View larger version (48K):
org.highwire.dtl.DTLVardef@33ca50org.highwire.dtl.DTLVardef@276f38org.highwire.dtl.DTLVardef@f6e66forg.highwire.dtl.DTLVardef@192b117_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIA new implant-less technique was used to reconstruct anterior cruciate ligament.
C_LIO_LIArtificial bone and fresh animal soft tissue used to simulate the fixation process.
C_LIO_LILoading condition were carefully chosen to simulate the post-operation.
C_LIO_LIComponents geometry had direct effect on biomechanical properties of the fixation.
C_LIO_LIOptimum geometry was found trough an experimental examination.
C_LI


title: Tree-based automated machine learning to predict biogas production for anaerobic co-digestion of organic waste

abstract: The dynamics of microbial communities involved in anaerobic digestion of mixed organic waste are notoriously complex and difficult to model, yet successful operation of anaerobic digestion is critical to the goals of diverting high-moisture organic waste from landfills. Machine learning (ML) is ideally suited to capturing complex and nonlinear behavior that cannot be modeled mechanistically. This study uses 8 years of data collected from an industrial-scale anaerobic co-digestion (AcoD) operation at a municipal wastewater treatment plant in Oakland, California, combined with a powerful automated ML method, Tree-based Pipeline Optimization Tool, to develop an improved understanding of how different waste inputs and operating conditions impact biogas yield. The model inputs included daily input volumes of 31 waste streams and 5 operating parameters. Because different wastes are broken down at varying rates, the model explored a range of time lags ascribed to each waste input ranging from 0 to 30 days. The results suggest that the waste types (including rendering waste, lactose, poultry waste, and fats, oils, and greases) differ considerably in their impact on biogas yield on both a per-gallon basis and a mass of volatile solids basis, while operating parameters are not useful predictors in a carefully operated facility.


title: Designing Interpretable Convolution-Based Hybrid Networks for Genomics

abstract: Hybrid networks that build upon convolutional layers with attention mechanisms have demon-strated improved performance relative to pure convolutional networks across many regulatory genome analysis tasks. Their inductive bias to learn long-range interactions provides an avenue to identify learned motif-motif interactions. For attention maps to be interpretable, the convolutional layer(s) must learn identifiable motifs. Here we systematically investigate the extent that architectural choices in convolution-based hybrid networks influence learned motif representations in first layer filters, as well as the reliability of their attribution maps generated by saliency analysis. We find that design principles previously identified in standard convolutional networks also generalize to hybrid networks. This work provides an avenue to narrow the spectrum of architectural choices when designing hybrid networks such that they are amenable to commonly used interpretability methods in genomics.


title: Single-cell multi-omic analysis of thymocyte development reveals NFAT as adriver of CD4/CD8 lineage commitment

abstract: CD4 and CD8 T cells play critical roles in the mammalian immune system. While their development within the thymus from the CD4+CD8+ stage has been widely studied as a model of lineage commitment, the underlying mechanism remains unclear. To deconstruct this process, we apply CITE-seq, measuring the transcriptome and over 100 surface proteins in thymocytes from wild-type and lineage-restricted mice. We jointly analyze the paired measurements to build a comprehensive timeline of RNA and protein expression in each lineage, supporting a sequential model of lineage determination in which both lineages go through an initial phase of CD4 lineage audition, which is followed by divergence and specification of CD8 lineage cells. We identify early differences implicating T cell receptor signaling via calcineurin-NFAT in driving CD4 lineage commitment. Pharmacological inhibition validates the requirement of calcineurin- NFAT for CD4, but not CD8, lineage development, providing insight into the CD4/CD8 commitment mechanism.


title: In vitro determinants of e-cig aerosol condensate bioavailability and toxicity: Influence of cell culture parameters and utility of a normalized dose metric in e-cig studies

abstract: Electronic cigarettes (e-cigs) have a strong foothold in the marketplace as a product to replace tobacco cigarette usage. Despite many researchers investigating the use of e-cigs and possible health issues, there is still controversy concerning how to evaluate and use e-cig condensates. Therefore, to identify factors that influence in vitro e-cig studies, we examined parameters that can impact experimental outcomes. We generated high wattage e-cig aerosol condensate (ECAC) to determine reproducible conditions to evaluate ECAC with respect to cellular survival. Cytotoxicity of ECAC was independent of serum conditions. However, cytotoxicity of ECAC is altered by treatment duration and by physical factors, including cell seeding density and volume of ECAC used. In addition, interactions between ECAC components and cells, as well as the culture vessel surface, diminish the bioavailability of ECAC components in vitro and altered the results obtained. Moreover, the cell seeding density changes reactive oxygen species production in response to ECAC exposure. Our data indicated that normalized ECAC doses (ECAC weight per cell) better reflect the toxicity of ECAC than nominal doses (ECAC percentage). These results provide factors for researchers to consider in the design of in vitro experiments using ECAC.


title: 2',3'-cAMP treatment mimics abiotic stress response

abstract: The role of the RNA degradation product 2,3-cyclic adenosine monophosphate (2,3-cAMP) is poorly understood. Recent studies have identified 2,3-cAMP in plant material and determined its role in stress signaling. The level of 2,3-cAMP increases upon wounding, dark, and heat, and 2,3-cAMP by binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insight into 2,3-cAMP function, we used a multi-omics approach combining transcriptomics, metabolomics, and proteomics to dissect Arabidopsis response to 2,3-cAMP treatment. We demonstrated that 2,3-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway from human cells might also exist in plants. Transcriptomic analysis revealed only minor overlap between 2,3-cAMP-and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2,3-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses including protein and RNA degradation products, glucosinolates, chaperones and SG components. Finally, we demonstrated that 2,3-cAMP treatment influences the movement of processing bodies, supporting the role of 2,3-cAMP in the formation and motility of membraneless organelles.


title: 2,3-cAMP treatment mimics stress molecular response in Arabidopsis thaliana


title: Electrophysiological correlates of proactive and reactive inhibition in a modified visual Go/NoGo task

abstract: Impulse control is crucial for everyday functioning in modern society. People with borderline personality disorder (BPD) suffer from impulse control impairment. According to the theory of dual mechanisms of control, motor impulse control can be divided into proactive and reactive modes. Proactive inhibition is involved before an event that might require inhibitory control. Reactive inhibition is initiated after the occurrence of an event that requires inhibitory control. Few studies have focused on proactive inhibition in relation to impaired impulse control, moreover electrophysiological evidence is scarce. Therefore, in the search for electrophysiological correlates of proactive and reactive inhibitions, we assessed event-related potentials elicited during a modified emotionally neutral visual Go/NoGo task in 28 clinically impulsive BPD patients and 35 healthy control (HC) subjects. In both groups, proactive inhibition was associated with enhanced late prestimulus activity and a suppressed poststimulus N2 component. In both groups, reactive inhibition was associated with enhanced poststimulus N2 and P3 components. We found no electrophysiological differences between HC subjects and BPD patients and both groups performed similarly in the task. Hence, the clinically observed impulse control impairment in BPD might act through different mechanisms other than altered inhibitory control in an emotionally neutral task.


title: A global dataset of seaweed net primary productivity

abstract: Net primary productivity (NPP) plays a pivotal role in the global carbon balance, but estimating the NPP of underwater habitats remains a challenging task. Seaweeds (marine macroalgae) form the largest and most productive underwater vegetated habitat on Earth. Yet, little is known about the distribution of their NPP at large spatial scales, despite more than 70 years of local-scale studies being scattered throughout the literature. We present a global dataset containing NPP records for 242 seaweed species at 419 individual sites distributed on all continents from the intertidal to 55 m depth. All records are standardized to annual aerial carbon production (g C m-2 yr-1) and are accompanied by detailed taxonomical and methodological information. The dataset presented here provides a basis for local, regional and global comparative studies of the NPP of underwater vegetation, and is pivotal for achieving a better understanding of the role seaweeds play in the global coastal carbon cycle.


title: Fifteen shades of clay: distinct microbial community profiles obtained from bentonite samples by cultivation and direct nucleic acid extraction

abstract: Characterizing the microbiology of swelling bentonite clays can help predict the long-term behaviour of deep geological repositories (DGRs), which are proposed as a solution for the management of used nuclear fuel worldwide. Such swelling clays represent an important component of several proposed engineered barrier system designs and, although cultivation-based assessments of bentonite clay are routinely conducted, direct nucleic acid detection from these materials has been difficult due to technical challenges. In this study, we generated direct comparisons of microbial abundance and diversity captured by cultivation and direct nucleic acid analyses using 15 reference bentonite clay samples. Regardless of clay starting material, the corresponding profiles from cultivation-based approaches were consistently associated with phylogenetically similar sulfate-reducing bacteria, denitrifiers, aerobic heterotrophs, and fermenters, demonstrating that any DGR-associated growth may be consistent, regardless of the specific bentonite clay starting material selected for its construction. Furthermore, dominant nucleic acid sequences in the as-received clay microbial profiles did not correspond with the bacteria that were enriched or isolated in culture. Few core taxa were shared among cultivation and direct nucleic acid analysis profiles, yet those in common were primarily affiliated with Streptomyces, Micrococcaceae, Bacillus, and Desulfosporosinus genera. These putative desiccation-resistant bacteria associated with diverse bentonite clay samples can serve as targets for experiments that evaluate microbial viability and growth within DGR-relevant conditions. Our data will be important for global nuclear waste management organizations, demonstrating that identifying appropriate design conditions with suitable clay swelling properties will prevent growth of the same subset of clay-associated bacteria, regardless of clay origin or processing conditions.


title: VirB4- and VirD4-like ATPases, components of a putative type 4C secretion system in Clostridioides difficile

abstract:
The type 4 secretion system (T4SS) represents a bacterial nanomachine capable of trans-cell wall transportation of proteins and DNA and which has attracted intense interest due to its roles in the pathogenesis of infectious diseases. During the current investigation we uncovered three distinct gene clusters in Clostridioides difficile strain 630 coding for proteins structurally related to components of the VirB4/D4 type 4C secretion system from Streptococcus suis strain 05ZYH33 and located within sequences of conjugative transposons (CTn). Phylogenic analysis shows that VirB4- and VirD4-like proteins of CTn4 locus, on one hand, and those of CTn2 and CTn5 loci, on the other hand, fit into separate clades, suggesting specific roles of identified secretion system variants in physiology of C. difficile. Our further study on VirB4- and VirD4-like products coded by CTn4 revealed that both proteins possess Mg2+-dependent ATPase activity, form oligomers (most probably, hexamers) in water solutions, and rely on potassium but not sodium ions for the highest catalytic rate. VirD4 binds nonspecifically to DNA and RNA. Its DNA binding activity strongly decreased with the W241A variant. Mutations in the nucleotide sequences coding for presumable Walker A and Walker B motifs decreased stability of the oligomers and significantly but not completely attenuated enzymatic activity of VirB4. In VirD4, substitutions of amino acid residues in the peptides reminiscent of Walker structural motifs resulted neither in attenuation of enzymatic activity of the protein nor influenced the oligomerization state of the ATPase.

ImportanceC. difficile is a Gram-positive, anaerobic, spore-forming bacterium that causes life-threatening colitis in humans. Major virulence factors of the microorganism include toxins TcdA, TcdB and CDT. However, other bacterial products, including a type 4C secretion system, have been hypothesized to contribute to the pathogenesis of the infection and are considered as possible virulence factors of C. difficile. In the current paper we describe structural organization of putative T4SS machinery in C. difficile and characterize its VirB4- and VirD4-like components. Our studies, in addition to significance for basic science, can potentially aid development of anti-virulence drugs suitable for treatment of C. difficile infection.


title: Specific phenotypic, genomic, and fitness evolutionary trajectories toward antibiotic resistance induced by pesticide co-stressors in Escherichia coli

abstract: To explore how co-occurring non-antibiotic environmental stressors affect evolutionary trajectories toward antibiotic resistance, we exposed susceptible Escherichia coli K-12 populations to environmentally relevant levels of pesticides and streptomycin for 500 generations. The coexposure substantially changed the phenotypic, genotypic, and fitness evolution trajectories, resulting in much stronger streptomycin resistance (>15-fold increase) of the populations. Antibiotic target modification mutations in rpsL and rsmG, which emerged and dominated at late stages of evolution, conferred the strong resistance even with less than 1% abundance, while the off-target mutations in nuoG, nuoL, glnE, and yaiW dominated at early stages only led to mild resistance (2.5 - 6-fold increase). Moreover, the strongly resistant mutants exhibited lower fitness costs even without the selective pressure and had lower minimal selection concentrations than the mildly resistant ones. Removal of the selective pressure did not reverse the strong resistance of coexposed populations at a later evolutionary stage. The findings suggest higher risks of the selection and propagation of strong antibiotic resistance in environments potentially impacted by antibiotics and pesticides.


title: Disentangling temporal associations in marine microbial networks

abstract: Microbial interactions are fundamental for Earths ecosystem functioning and biogeochemical cycling. Nevertheless, they are challenging to identify and remain barely known. The omics-based censuses are helpful to predict microbial interactions through the inference of static association networks. However, since microbial interactions are highly dynamic, we have developed a post-network-construction approach to generate a temporal network from a single static network. We applied it to understand the monthly microbial associations dynamics occurring over ten years in the Blanes Bay Microbial Observatory (Mediterranean Sea). For the decade, we identified persistent, seasonal, and temporary microbial associations. Moreover, we found that the temporal network appears to follow an annual cycle, collapsing and reassembling when transiting between colder and warmer waters. We observed higher repeatability in colder than warmer months. Altogether, our results indicate that marine microbial networks follow recurrent temporal dynamics, which need to be accounted to better understand the dynamics of the ocean microbiome.


title: Predicting protein domain temperature adaptation across the prokaryote-eukaryote divide

abstract: Protein thermostability is important for fitness but difficult to measure across the proteome. Fortunately, protein thermostability is correlated with prokaryote optimal growth temperatures (OGTs), which can be predicted from genome features. Models that can predict temperature sensitivity across the prokaryote-eukaryote divide would help inform how eukaryotes adapt to elevated temperatures, such as those predicted by climate change models. In this study we test whether prediction models can cross the prokaryote-eukaryote divide to predict protein stability in both prokaryotes and eukaryotes. We compare models built using a) the whole proteome, b) Pfam domains, and c) individual amino acid residues. Proteome-wide models accurately predict prokaryote optimal growth temperatures (r2 up to 0.93), while site-specific models demonstrate that nearly half of the proteome is associated with optimal growth temperature in both Archaea and Bacteria. Comparisons with the small number of eukaryotes with temperature sensitivity data suggest that site-specific models are the most transferable across the prokaryote-eukaryote divide. Using the site-specific models, we evaluated temperature sensitivity for 323,850 amino acid residues in 2,088 Pfam domain clusters in Archaea and Bacteria species separately. 59.0% of tested residues are significantly associated with OGT in Archaea and 75.2% of tested residues are significantly associated with OGT in Bacteria species at a 5% false discovery rate. These models make it possible to identify which Pfam domains and amino acid residues are involved in temperature adaptation and facilitate future research questions about how species will fare in the face of increasing environmental temperatures.


title: Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

abstract: High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3-OH group of the primer strand to the -phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.


title: Calorie Restriction Maintains Mitochondrial Function and Redox Balance Avoiding Lipidomic Reprogramming during Isoproterenol-Induced Cardiac Hypertrophy

abstract: Cardiac hypertrophy induces a metabolic shift, leading to a preferential consumption of glucose (over fatty acids) to support the high energetic demand. Typically, health cardiac tissue utilizes more fat than any other organ. Calorie restriction is a dietary procedure that induces health benefits and lifespan extension in many organisms. Given the beneficial effects of calorie restriction and the metabolic dysregulation seen during cardiac hypertrophy, we hypothesized that calorie restriction prevents cardiac hypertrophy, lipid, mitochondrial, and redox dysregulations. Strikingly, calorie restriction reversed isoproterenol-induced cardiac hypertrophy, lowered succinate driven mitochondrial H2O2 production, improved mitochondrial function (indicated as a higher Respiratory Control Ratio - RCR) and avoided mitochondrial superoxide dismutase (MnSOD) and glutathione peroxidase (GPX) repression. To gain insight into how calorie restriction could interfere with the metabolic changes induced by cardiac hypertrophy, we performed lipidomic profiling. Calorie restriction protected against the consumption of several triglycerides (TG) linked to unsaturated fatty acids, and the accumulation of TGs containing saturated fatty acids observed in hypertrophic samples. Cardiac hypertrophy induced an increase in ceramides, phosphoethanolamines and acylcarnitines (12:0, 14:0, 16:0 and 18:0) that were also reversed by calorie restriction. Altogether, our data demonstrate that hypertrophy changes the cardiac lipidome, causes mitochondrial disturbances and oxidative stress. All these changes are prevented by calorie restriction intervention in vivo. This study uncovers calorie restriction as a resource protect cardiac tissue and prevent cardiac hypertrophy-induced lipidomic remodeling.


title: Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 reveal a novel binding site for positive modulators

abstract: Kv3 ion-channels constitute a class of functionally distinct voltage gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 [A] resolution using full length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well resolved Zinc site and displaying a rotation by 35{degrees}. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.


title: Electronic "photoreceptors" enable prosthetic vision with acuity matching the natural resolution in rats

abstract: Localized stimulation of the inner retinal neurons for high-acuity prosthetic vision requires small pixels and minimal cross-talk from neighboring electrodes. Local return electrodes within each pixel limit crosstalk, but can over-constrain the electric field, thus precluding efficient stimulation with subretinal pixels smaller than 50 m. Here we demonstrate high-resolution prosthetic vision based on a novel design of a photovoltaic array, where field confinement is achieved dynamically, leveraging the adjustable conductivity of the diodes under forward bias to turn the designated pixels into transient returns. We validated computational modeling of the field confinement in such an optically-controlled circuit by ex-vivo and in-vivo measurements. Most importantly, using this strategy, we demonstrated that the grating acuity with 40 m pixels matches the pixel pitch, while with 20 m pixels, it reaches the 28 m limit of the natural visual resolution in rats. This method enables customized field shaping based on individual retinal thickness and distance from the implant, paving the way to prosthetic vision with acuity as high as 20/80 in atrophic macular degeneration.


title: Interaction between SARS-CoV-2 spike glycoprotein and human skin models: a molecular dynamics study

abstract: The possibility of contamination of human skin by infectious virions plays an important role in indirect transmission of respiratory viruses but little is known about the fundamental physico-chemical aspects of the virus-skin interactions. In the case of coronaviruses, the interaction with surfaces (including the skin surface) is mediated by their large glycoprotein spikes that protrude from (and cover) the viral envelope. Here, we perform all atomic simulations between the SARS-CoV-2 spike glycoprotein and human skin models. We consider an "oily" skin covered by sebum and a "clean" skin exposing the stratum corneum. The simulations show that the spike tries to maximize the contacts with stratum corneum lipids, particularly ceramides, with substantial hydrogen bonding. In the case of "oily" skin, the spike is able to retain its structure, orientation and hydration over sebum with little interaction with sebum components. Comparison of these results with our previous simulations of the interaction of SARS-CoV-2 spike with hydrophilic and hydrophobic solid surfaces, suggests that the"soft" or "hard" nature of the surface plays an essential role in the interaction of the spike protein with materials.


title: The role of reading experience in atypical cortical tracking of speech and speech-in-noise in dyslexia

abstract:
Dyslexia is a frequent developmental disorder in which reading acquisition is delayed and that is usually associated with difficulties understanding speech in noise. At the neuronal level, children with dyslexia were reported to display abnormal cortical tracking of speech (CTS) at phrasal rate. Here, we aimed to determine if abnormal tracking is a cause or a consequence of dyslexia and if it is modulated by the severity of dyslexia or the presence of acoustic noise.

We included 26 school-age children with dyslexia, 26 age-matched controls and 26 reading-level matched controls. All were native French speakers. Childrens brain activity was recorded with magnetoencephalography while they listened to continuous speech in noiseless and multiple noise conditions. CTS values were compared between groups, conditions and hemispheres, and also within groups, between children with best and worse reading performance.

Syllabic CTS was significantly reduced in the right superior temporal gyrus in children with dyslexia compared with controls matched for age but not for reading level. Among children with dyslexia, phrasal CTS tended to lateralize to the left hemisphere in severe dyslexia and lateralized to the right hemisphere in children with mild dyslexia and in all control groups. Finally, phrasal CTS was lower in children with dyslexia compared with age-matched controls, but only in informational noise conditions. No such effect was seen in comparison with reading-level matched controls.

Overall, our results confirmed the finding of altered neuronal basis of speech perception in noiseless and babble noise conditions in dyslexia compared with age-matched peers. However, the absence of alteration in comparison with reading-level matched controls suggests that such alterations are a consequence of reduced reading experience rather than a cause of dyslexia.

HighlightsO_LIThe cortical tracking of speech (CTS) was assessed in dyslexia and proper controls
C_LIO_LIPhrasal CTS and its resistance to noise were altered in dyslexia
C_LIO_LISuch alterations were not found in comparison with controls matched for reading level
C_LIO_LIThe severity of dyslexia modulated the hemispheric lateralization of phrasal CTS
C_LI


title: Gauging the capacity for concurrent motor planning in human fMRI with information constraints in bounded rational decision-making models

abstract: While traditional theories of sensorimotor processing have often assumed a serial decision-making pipeline, more recent approaches have suggested that multiple actions may be planned concurrently and vie for execution. Evidence for the latter almost exclusively stems from electrophysiological studies in monkeys. We here study concurrent prospective motor planning in humans by recording functional magnetic resonance imaging (fMRI) during a delayed response task engaging movement sequences towards multiple potential targets. We find that delay activity in posterior parietal cortex, frontal cortex and the cerebellum modulates both with sequence complexity and the number of potential targets. We tested the hypothesis that this modulation is best explained by concurrent prospective planning as opposed to the mere maintenance of potential targets in memory. We devise a bounded rationality model with information constraints that optimally assigns information resources for planning and memory for this task and determine predicted information profiles according to the two hypotheses. When regressing delay activity on these model predictions, we find that the concurrent prospective planning strategy provides a significantly better explanation of the fMRI-signal modulations. Moreover, we find that concurrent prospective planning is more costly and thus limited for most subjects, as expressed by the best fitting information capacities. We conclude that bounded rational decision-making models allow relating both behavior and neural representations to utilitarian task descriptions based on bounded optimal information-processing assumptions.


title: Bounded rational decision-making models suggest capacity-limited concurrent motor planning in human posterior parietal and frontal cortex


title: The Burkholderia cenocepacia type VI secretion system effector TecA is a virulence factor during lung infection

abstract:
Burkholderia cenocepacia (Bc) is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. Bc is strongly associated with cepacia syndrome making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of Bc in lung infections and cepacia syndrome remain to be uncovered. Bc is primarily an intracellular pathogen, and encodes the type VI secretion system (T6SS) anti-host effector TecA, which is translocated into host cells. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines such as IL-1{beta} from macrophages. Previous work with the Bc clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second Bc clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and CF mice. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a Bc virulence factor that exacerbates lung inflammation, weight loss, and lethality in a mouse lung infection model.

ImportanceBc is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (Loutet and Valvano 2010). Prior to this study virulence factors of Bc important for causing lethal disease had not been identified in a CF animal model of lung infection. Results of this study describe a CF mouse model and its use in demonstrating that the T6SS effector TecA of Bc exacerbates inflammatory cell recruitment and weight loss and is required for lethality and thus acts as a key virulence factor during lung infection. This model will be important in further studies to better understand TecAs role as a virulence factor and in investigating ways to prevent or treat Bc infections in CF patients. Additionally, TecA may be the founding member of a family of virulence factors in opportunistic pathogens.


title: A genetic toolkit for investigating Clavibacter: markerless deletion, permissive site identification and an integrative plasmid

abstract: The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advancements of these techniques in Gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens which cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen C. michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and confirmed using whole genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a non-replicating integrative plasmid that enables recombination into a permissive genomic location. Expression of eGFP by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advancements for investigating Gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.