title: Age-related demethylation of the TDP-43 autoregulatory region in the human motor cortex

abstract: In amyotrophic lateral sclerosis (ALS), TAR DNA-binding protein 43 (TDP-43), which is encoded by TARDBP, forms aggregates in the motor cortex. This aggregate formation may be triggered by an increase in the TDP-43 level with aging. However, the amount of TDP-43 is autoregulated by alternative splicing of the TARDBP 3UTR, and how this autoregulation is affected by aging remains to be elucidated. We found that DNA demethylation in the autoregulatory region in the TARDBP 3'UTR reduced alternative splicing and increased TARDBP mRNA expression. Furthermore, in the human motor cortex, we found that this region was demethylated with aging, resulting in increased expression of TARDBP mRNA. The acceleration of DNA demethylation in the motor cortex was associated with the age of ALS onset. In summary, the dysregulation of TDP-43 autoregulation by age-related DNA demethylation in the motor cortex may explain the contribution of aging and motor system selectivity in ALS.


title: Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

abstract: Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic integrity in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based test for the detection of hotspot mutations test. Cell-line identity was authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by qPCR for progenitor, neuronal, cortical, and glial markers. Taken together, we present a multistep quality control workflow to evaluate variability and reproducibility across and between iPSCs.

category: cell biology


title: Mapping structure to function and behavior with individual-level connectome embedding

abstract: The connectome, a comprehensive map of the brains anatomical connections, is often summarized as a matrix comprising all dyadic connections among pairs of brain regions. This representation cannot capture higher-order relations within the brain graph. Connectome embedding (CE) addresses this limitation by creating compact vectorized representations of brain nodes capturing their context in the global network topology. Here, nodes "context" is defined as random walks on the brain graph and as such, represents a generative model of diffusive communication around nodes. Applied to group-averaged structural connectivity, CE was previously shown to capture relations between inter-hemispheric homologous brain regions and uncover putative missing edges from the network reconstruction. Here we extend this framework to explore individual differences with a novel embedding alignment approach. We test this approach in two lifespan datasets (NKI: n=542; Cam-CAN: n=601) that include diffusion-weighted imaging, resting-state fMRI, demographics and behavioral measures. We demonstrate that modeling functional connectivity with CE substantially improves structural to functional connectivity mapping both at the group and subject level. Furthermore, age-related differences in this structure-function mapping are preserved and enhanced. Importantly, CE captures individual differences by out-of-sample prediction of age and intelligence. The resulting predictive accuracy was higher compared to using structural connectivity and functional connectivity. We attribute these findings to the capacity of the CE to incorporate aspects of both anatomy (the structural graph) and function (diffusive communication). Our novel approach allows mapping individual differences in the connectome through structure to function and behavior.

category: neuroscience


title: Mapping individual differences across brain network structure to function and behavior with connectome embedding


title: An integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation

abstract: Protein tyrosine phosphatase: phospho-protein complex structure determination, which requires to understand how specificity is achieved at the protein level remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTP{varepsilon}D1 and phospho-SrcKD as a model system, we established an integrative workflow involving protein crystallography, SAXS and pTyr-tailored MD simulations to reveal the complex formed between rPTP{varepsilon}D1 and phospho-SrcKD, revealing transient protein-protein interactions distal to the active site. To support our finding, we determined the associate rate between rPTP{varepsilon}D1 and phospho-SrcKD and showed that a single mutation on rPTP{varepsilon}D1 disrupts this transient interaction, resulting in the reduction of association rate and activity. Our simulations suggest that rPTP{varepsilon}D1 employs a binding mechanism involving conformational change prior to the engagement of cSrcKD. This integrative approach is applicable to other PTP: phospho-protein complex determination and is a general approach for elucidating transient protein surface interactions.

category: biophysics


title: Lymphotoxin β receptor: A crucial role in innate and adaptive immune responses against Toxoplasma gondii

abstract: The LT{beta}R plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LT{beta}R is crucial for surviving acute toxoplasmosis, however, up to now a functional analysis is largely incomplete. Here, we demonstrate that the LT{beta}R is a key regulator required for the intricate balance of adaptive immune responses. T. gondii infected LT{beta}R-/- mice show globally altered IFN{gamma} regulation, reduced IFN{gamma}-controlled host effector molecule expression, impaired T cell functionality and an absent anti-parasite specific IgG response resulting in a severe loss of immune control of the parasites. Reconstitution of LT{beta}R-/- mice with toxoplasma immune serum significantly prolongs the survival following T. gondii infection. Notably, analysis of RNAseq data clearly indicates a specific effect of T. gondii infection on the B cell response and isotype switching. This study unfolds the decisive role of the LT{beta}R in cytokine regulation and adaptive immune responses to control T. gondii.

category: immunology


title: High-throughput Identification of Eukaryotic Parasites and Arboviruses in Mosquitoes

abstract: Vector-borne pathogens cause many human infectious diseases and are responsible for high mortality and morbidity throughout the world. They can also cause livestock epidemics with dramatic social and economic consequences. Due to the high costs, vector-borne disease surveillance is often limited to current threats, and the investigation of emerging pathogens typically occur after the reports of clinical cases. Here, we use high-throughput sequencing to detect and identify a wide range of parasites and viruses carried by mosquitoes from Cambodia, Guinea, Mali and Maryland. We apply this approach to individual Anopheles mosquitoes as well as pools of mosquitoes captured in traps; and compare the outcomes of this assay when applied to DNA or RNA. We identified known human and animal pathogens and mosquito parasites belonging to a wide range of taxa, insect Flaviviruses, and novel DNA sequences from previously uncharacterized organisms. Our results also revealed that analysis of the content of an entire trap is an efficient approach to monitor and identify potential vector-borne pathogens in large surveillance studies, and that analyses of RNA extracted from mosquitoes is preferable, when possible, over DNA-based analyses. Overall, we describe a flexible and easy-to-customize assay that can provide important information for vector-borne disease surveillance and research studies to efficiently complement current approaches.

category: microbiology


title: Cutibacterium acnes clonal complexes display various growth rates in blood culture bottles used for diagnosing orthopedic device related infections

abstract:
Blood-culture bottles (BCBs) are widely used to improve the diagnosis of orthopedic device-related infections. Data is scarce on the growth of Cutibacterium acnes and its genotypes in BCBs under real-life clinical conditions.

We studied 39 cases of revision arthroplasty for which at least one intraoperative sample yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F [Lytic Ana]) and/or solid media. Genotyping of C. acnes isolates from the 39 cases allowed: i) the identification of 49 non-redundant isolates belonging to four clonal complexes (CCs): CC18, CC28, CC36, and CC53 and ii) the determination of infectant and contaminant strains. Under real-life clinical conditions, Lytic Ana alone was more often positive for contaminants than infectant strains (18/36 [50%] versus 2/13 [15.4%]; p = 0.047). The time to detection (TTD) values in Lytic Ana were shorter for CC53 than other CCs (mean [SD] TTD: 77 [15] versus 165 [71] hours; p = 0.02). CC53 was confirmed to grow faster than other CCs by studying an enlarged panel of 70 genotyped C. acnes strains inoculated in vitro into Lytic Ana vials (mean [SD] TTD: 73 [13] versus 122 [50] hours; p < 0.001).

The use of Lytic Ana BCBs in orthopedics increases the recovery rate of C. acnes but leads to the isolation of proportionally more contaminants than true infectant strains. TTD values are much shorter for CC53 strains, irrespective of their being infectant or contaminant. TTD does not solely reflect the bacterial load of samples but also clonal complex-related traits.


title: Genes encoding teleost orthologs of human haplo-insufficient and monoallelic genes remain in duplicate more frequently than the whole genome

abstract: Gene dosage is important is an important issue both in cell and evolutionary biology. Most genes are present in two copies in eukaryotic cells. The first outstanding exception is monoallelic gene expression (MA) that concerns genes localized on the X chromosome or in regions undergoing parental imprinting in eutherians, and many other genes scattered throughout the genome. The second exception concerns haploinsufficiency (HI), responsible for the fact that a single functional copy of a gene in a diploid organism is insufficient to ensure a normal biological function. One of the most important mechanisms ensuring functional innovation during evolution is Whole genome duplication (WGD). In addition to the two WGDs that have occurred in vertebrate genomes, the teleost genomes underwent an additional WGD, after their divergence from tetrapod. In the present work, we have studied on 57 teleost species whether the orthologs of human MA or HI genes remain more frequently in duplicates or returned more frequently in singleton than the rest of the genome. Our results show that the teleost orthologs of HI human genes remained more frequently in duplicate than the rest of the genome in all the teleost species studied. No signal was observed for the orthologs of genes localized on the human X chromosome or subjected to parental imprinting. Surprisingly, the teleost orthologs of the other human MA genes remained in duplicate more frequently than the rest of the genome for most teleost species. These results suggest that the teleost orthologs of MA and HI human genes also undergo selective pressures either related to absolute protein amounts and/or of dosage balance issues. However, these constraints seem to be different for MA genes in teleost in comparison with human genomes.

category: evolutionary biology


title: Functional annotation of human long noncoding RNAs using chromatin conformation data

abstract: Transcription of the human genome yields mostly long non-coding RNAs (lncRNAs). Systematic functional annotation of lncRNAs is challenging due to their low expression level, cell type-specific occurrence, poor sequence conservation between orthologs, and lack of information about RNA domains. Currently, 95% of human lncRNAs have no functional characterization. Using chromatin conformation and Cap Analysis of Gene Expression (CAGE) data in 18 human cell types, we systematically located genomic regions in spatial proximity to lncRNA genes and identified functional clusters of interacting protein-coding genes, lncRNAs and enhancers. Using these clusters we provide a cell type-specific functional annotation for 7,651 out of 14,198 (53.88%) lncRNAs. LncRNAs tend to have specialized roles in the cell type in which it is first expressed, and to incorporate more general functions as its expression is acquired by multiple cell types during evolution. By analyzing RNA-binding protein and RNA-chromatin interaction data in the context of the spatial genomic interaction map, we explored mechanisms by which these lncRNAs can act.

category: genomics


title: NeurotoxKb: compilation, curation and exploration of a knowledgebase of environmental neurotoxicants specific to mammals

abstract: Exposure to environmental neurotoxicants is a significant concern due to their potential to cause permanent or irreversible damage to the human nervous system. Here, we present the first dedicated knowledgebase, NeurotoxKb, on environmental neurotoxicants specific to mammals. Using a detailed workflow, we have compiled 475 potential non-biogenic neurotoxicants from 835 published studies with evidence of neurotoxicity specific to mammals. A unique feature of NeurotoxKb is the manual curation effort to compile and standardize the observed neurotoxic effects for the potential neurotoxicants from 835 published studies. For the 475 potential neurotoxicants, we have compiled diverse information such as chemical structures, environmental sources, chemical classification, physicochemical properties, molecular descriptors, predicted ADMET properties, and target human genes. To better understand the prospect of human exposure, we have explored the presence of potential neurotoxicants in external exposomes via two different analyses. By analyzing 55 chemical lists representing global regulations and guidelines, we reveal potential neurotoxicants both in regular use and produced in high volume. By analyzing human biospecimens, we reveal potential neurotoxicants detected in them. Lastly, a construction of the chemical similarity network and ensuing analysis revealed the diversity of the toxicological space of 475 potential neurotoxicants. NeurotoxKb is accessible online at: https://cb.imsc.res.in/neurotoxkb/.

category: pharmacology and toxicology


title: An Open-Source Pipeline for Analyzing Changes In Microglial Morphology

abstract: Changes in microglial morphology are powerful indicators of the inflammatory state of the brain. Here we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and "inflammation" training data and uses dimensionality reduction to generate a single metric of morphological change (an  inflammation index). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.


title: Ecosystem engineering alters density-dependent feedbacks in an aquatic insect population

abstract: Ecosystem engineers have large impacts on the communities in which they live, and these impacts may feed back to populations of engineers themselves. In this study, we assessed the effect of ecosystem engineering on density-dependent feedbacks for midges in Lake Myvatn, Iceland. The midge larvae reside in the sediment and build silk tubes that provide a substrate for algal growth, thereby elevating benthic primary production. Benthic algae are in turn the primary food source for the midge larvae, setting the stage for the effects of engineering to feed back to the midges themselves. Using a field mesocosm experiment manipulating larval midge densities, we found a generally positive but nonlinear relationship between density and benthic production. Furthermore, adult emergence increased with the primary production per midge larva. By combining these two relationships in a simple model, we found that the positive effect of midges on benthic production weakened the negative density dependence at low to intermediate larval densities. However, this benefit disappeared at high densities when midge consumption of primary producers exceeded their positive effects on primary production through ecosystem engineering. Our results illustrate how ecosystem engineering can alter density-dependent feedbacks for engineer populations.

category: ecology


title: Does the punishment fit the crime? Consequences and diagnosis of misspecified detection functions in Bayesian spatial capture-recapture modelling

abstract:
O_LISpatial capture-recapture (SCR) is now used widely to estimate wildlife densities. At the core of SCR models lies the detection function, linking individual detection probability to the distance from its latent activity center. The most common function (half-normal) assumes a bivariate normal space use and consequently detection pattern. This is likely an oversimplification and misrepresentation of real-life animal space use patterns, but studies have reported that density estimates are relatively robust to misspecified detection functions. However, information about consequences of such misspecification on space use parameters (e.g. home range area), as well as diagnostic tools to reveal it are lacking.
C_LIO_LIWe simulated SCR data under six different detection functions, including the half-normal, to represent a wide range of space use patterns. We then fit three different SCR models, with the three simplest detection functions (half-normal, exponential and half-normal plateau) to each simulated data set. We evaluated the consequences of misspecification in terms of bias, precision and coverage probability of density and home range area estimates. We also calculated Bayesian p-values with respect to different discrepancy metrics to assess whether these can help identify misspecifications of the detection function.
C_LIO_LIWe corroborate previous findings that density estimates are robust to misspecifications of the detection function. However, estimates of home range area are prone to bias when the detection function is misspecified. When fitted with the half-normal model, average relative bias of 95% kernel home range area estimates ranged between -25% and 26% depending on the misspecification. In contrast, the half-normal plateau model (an extension of the half-normal) returned average relative bias that ranged between -26% and -4%. Additionally, we found useful heuristic patterns in Bayesian p-values to diagnose the misspecification in detection function.
C_LIO_LIOur analytical framework and diagnostic tools may help users select a detection function when analyzing empirical data, especially when space use parameters (such as home range area) are of interest. We urge development of additional custom goodness of fit diagnostics for Bayesian SCR models to help practitioners identify a wider range of model misspecifications.
C_LI


title: Quantifying community responses to environmental variation from replicate time series

abstract: Time-series data for ecological communities are increasingly available from long-term studies designed to track species responses to environmental change. However, classical multivariate methods for analyzing community composition have limited applicability for time series, as they do not account for temporal autocorrelation in community-member abundances. Furthermore, traditional approaches often obscure the connections between responses at the community level and those for individual taxa, limiting their capacity to infer mechanisms of community change. We show how linear mixed models that account for group-specific temporal autocorrelation and observation error can be used to infer both taxon- and community-level responses to environmental predictors from replicated time-series data. Variation in taxon-specific responses to predictors is modeled using random effects, which can be used to characterize variation in community composition. Moreover, the degree of autocorrelation is estimated separately for each taxon, since this is likely to vary due to differences in their underlying population dynamics. We illustrate the utility of the approach by analyzing the response of a predatory arthropod community to spatiotemporal variation in allochthonous resources in a subarctic landscape. Our results show how mixed models with temporal autocorrelation provide a unified approach to characterizing taxon- and community-level responses to environmental variation through time.


title: Low generalizability of polygenic scores in African populations due to genetic and environmental diversity

abstract: African populations are vastly underrepresented in genetic studies but have the most genetic variation and face wide-ranging environmental exposures globally. Because systematic evaluations of genetic prediction had not yet been conducted in ancestries that span African diversity, we calculated polygenic risk scores (PRS) in simulations across Africa and in empirical data from South Africa, Uganda, and the UK to better understand the generalizability of genetic studies. PRS accuracy improves with ancestry-matched discovery cohorts more than from ancestry-mismatched studies. Within ancestrally and ethnically diverse South Africans, we find that PRS accuracy is low for all traits but varies across groups. Differences in African ancestries contribute more to variability in PRS accuracy than other large cohort differences considered between individuals in the UK versus Uganda. We computed PRS in African ancestry populations using existing European-only versus ancestrally diverse genetic studies; the increased diversity produced the largest accuracy gains for hemoglobin concentration and white blood cell count, reflecting large-effect ancestry-enriched variants in genes known to influence sickle cell anemia and the allergic response, respectively. Differences in PRS accuracy across African ancestries originating from diverse regions are as large as across out-of-Africa continental ancestries, requiring commensurate nuance.

category: genetics


title: Comparing utility functions between risky and riskless choice in rhesus monkeys

abstract: Decisions can be risky or riskless, depending on the outcomes of the choice. Expected Utility Theory describes risky choices as a utility maximization process: we choose the option with the highest subjective value (utility), which we compute considering both the options value and its associated risk. According to the random utility maximization framework, riskless choices could also be based on a utility measure. Neuronal mechanisms of utility-based choice may thus be common to both risky and riskless choices. This assumption would require the existence of a utility function that accounts for both risky and riskless decisions. Here, we investigated whether the choice behavior of macaque monkeys in riskless and risky decisions could be described by a common underlying utility function. We found that the utility functions elicited in the two choice scenarios were different from each other, even after taking into account the contribution of subjective probability weighting. Our results suggest that distinct utility representations exist for riskless and risky choices, which could reflect distinct neuronal representations of the utility quantities, or distinct brain mechanisms for risky and riskless choices. The different utility functions should be taken into account in neuronal investigations of utility-based choice.

category: animal behavior and cognition


title: Dimensionality reduction by UMAP reinforces sample heterogeneity analysis in bulk transcriptomic data

abstract: Transcriptome profiling and differential gene expression constitute a ubiquitous tool in biomedical research and clinical application. Linear dimensionality reduction methods especially principal component analysis (PCA) are widely used in detecting sample-to-sample heterogeneity in bulk transcriptomic datasets so that appropriate analytic methods can be used to correct batch effects, remove outliers and distinguish subgroups. In response to the challenge in analysing transcriptomic datasets with large sample size such as single-cell RNA-sequencing (scRNA-seq), non-linear dimensionality reduction methods were developed. t-distributed stochastic neighbour embedding (t-SNE) and uniform manifold approximation and projection (UMAP) show the advantage of preserving local information among samples and enable effective identification of heterogeneity and efficient organisation of clusters in scRNA-seq analysis. However, the utility of t-SNE and UMAP in bulk transcriptomic analysis has not been carefully examined. Therefore, we compared major dimensionality reduction methods (linear: PCA; nonlinear: multidimensional scaling (MDS), t-SNE, and UMAP) in analysing 71 bulk transcriptomic datasets with large sample sizes. UMAP was found superior in preserving sample level neighbourhood information and maintaining clustering accuracy, thus conspicuously differentiating batch effects, identifying pre-defined biological groups and revealing in-depth clustering structures. We further verified that new clustering structures visualised by UMAP were associated with biological features and clinical meaning. Therefore, we recommend the adoption of UMAP in visualising and analysing of sizable bulk transcriptomic datasets.

category: bioinformatics


title: DISMIR: a deep learning-based cancer-detection method by integrating DNA sequence and methylation information of individual cell-free DNA reads

abstract: Detecting cancer signals in cell-free DNA (cfDNA) high-throughput sequencing data is emerging as a novel non-invasive cancer detection method. Due to the high cost of sequencing, it is crucial to make robust and precise prediction with low-depth cfDNA sequencing data. Here we propose a novel approach named DISMIR, which can provide ultrasensitive and robust cancer detection by integrating DNA sequence and methylation information in plasma cfDNA whole genome bisulfite sequencing (WGBS) data. DISMIR introduces a new feature termed as "switching region" to define cancer-specific differentially methylated regions, which can enrich the cancer-related signal at read-resolution. DISMIR applies a deep learning model to predict the source of every single read based on its DNA sequence and methylation state, and then predicts the risk that the plasma donor is suffering from cancer. DISMIR exhibited high accuracy and robustness on hepatocellular carcinoma detection by plasma cfDNA WGBS data even at ultra-low sequencing depths. Analysis showed that DISMIR tends to be insensitive to alterations of single CpG sites methylation states, which suggests DISMIR could resist to technical noise of WGBS. All these results showed DISMIR with the potential to be a precise and robust method for low-cost early cancer detection.


title: A Unified Probabilistic Modeling Framework for Eukaryotic Transcription Based on Nascent RNA Sequencing Data

abstract: Nascent RNA sequencing protocols, such as PRO-seq and NET-seq, are now widely used in the study of eukaryotic transcription, and these experimental techniques have given rise to a variety of statistical and machine-learning methods for data analysis. These computational methods, however, are generally designed to address specialized signal-processing or prediction tasks, rather than directly describing the dynamics of RNA polymerases as they move along the DNA template. Here, I introduce a general probabilistic model that describes the kinetics of transcription initiation, elongation, pause release, and termination, as well as the generation of sequencing read counts. I show that this generative model enables estimation of separate pause-release rates, termination rates, and the initiation/elongation rate ratio up to a proportionality constant. Furthermore, if applied to time-course data in a nonequilibrium setting, the model can be used to estimate elongation rates. This model leads naturally to likelihood ratio tests for differences between genes, conditions, or species in various rates of interest. If read counts are assumed to be Poisson-distributed, convenient, closed-form solutions are available for both parameter estimates and likelihood-ratio-test statistics. Straightforward extensions of the model accommodate uncertainty in the pause site and steric hindrance of initiation by paused polymerases. Additional extensions address Bayesian inference under the Poisson model and a generalized linear model that can be used to discover genomic features associated with rates of elongation. Finally, I address technicalities concerning estimation of library size, normalization and sequencing replicates. Altogether, this modeling framework enables a unified treatment of many common tasks in the analysis of nascent RNA sequencing data.


title: MiDAS - Meaningful Immunogenetic Data at Scale

abstract: Human immunogenetic variation in the form of HLA and KIR types has been shown to be strongly associated with a multitude of immune-related phenotypes. We present MiDAS, an R package enabling statistical association analysis and using immunogenetic data transformation functions for HLA amino acid fine mapping, analysis of HLA evolutionary divergence as well as HLA-KIR interactions. MiDAS closes the gap between inference of immunogenetic variation and its efficient utilization to make meaningful discoveries.


title: Exposure to fruit-flavoring during adolescence increases nicotine consumption and promotes dose escalation

abstract: The rise of e-cigarette popularity has sparked interest in the role of palatable flavors on nicotine use. Despite growing evidence that sweet flavorants enhance nicotine reward, their influence on nicotine consumption has not been studied extensively. In addition, the impact that flavored nicotine use in adolescence could have on nicotine reward and dependence in adulthood remains unclear. This study examined the role of flavored nicotine access on nicotine preference and consumption longitudinally, from adolescence to adulthood. Male and female adolescent mice preferred a fruit-flavored nicotine solution. However, only adolescent female mice with access to flavored nicotine consumed higher doses. Furthermore, while adolescent male mice escalated consumption of both flavored and unflavored nicotine, female mice only escalated when given access to flavored nicotine. As mice matured into adulthood, there was no evidence that a history of flavored-nicotine access altered preference for unflavored nicotine. However, when the nicotine concentration was reduced, mice that had consumed strawberry-flavored nicotine in adolescence maintained baseline nicotine consumption levels longer than mice that initiated nicotine use without flavor in adolescence. Finally, addition of fruit-flavorants into the nicotine solution during adulthood led to increased levels of nicotine consumption, regardless of previous flavored-nicotine access or of familiarity with the selected flavorant. These results indicate that flavorants increase nicotine consumption independent of life stage, possibly posing a disproportionate risk to adolescent females. Our results also point to an effect of adolescent flavored-nicotine use on nicotine dose maintenance in adulthood, which could have implications for the success of future quit attempts.


title: Dominant and nondominant distal radius microstructure: predictors of asymmetry and effects of a unilateral mechanical loading intervention

abstract: Most information about distal radius microstructure is based on the non-dominant forearm, with little known about the factors that contribute to bilateral asymmetries in the general population, or what factors may influence bilateral changes over time. Here, we analyzed bilateral longitudinal high resolution peripheral quantitative computed tomography (HRpQCT) data collected over a 12-month period as part of a clinical trial that prescribed a well-controlled, compressive loading task to the nondominant forearm. Baseline data from 102 women age 21-40, and longitudinal data from 66 women who completed the 12-month trial, were examined to determine factors responsible for side-to-side asymmetries in bone structure and change in structure over time. Cross-sectionally, the dominant radius had 2.4%-2.7% larger cross-sectional area, trabecular area, and bone mineral content than the nondominant radius, but no other differences were noted. Those who more strongly favored their dominant arm had significantly more, thinner, closely spaced trabecular struts in their dominant versus nondominant radius. Individuals assigned to a loading intervention had significant bilateral gains in total bone mineral density (2.0% and 1.2% in the nondominant versus dominan sides), and unilateral gains in cortical area (3.1%), thickness (3.0%), bone mineral density (1.7%) and inner trabecular density (1.3%). Each of these gains were significantly predicted by loading dose, a metric that included bone strain, number of cycles, and strain rate. Within individuals, change was negatively associated with age, meaning that women closer to age 40 experienced less of a gain in bone versus those closer to age 21. We believe that dominant/nondominant asymmetries in bone structure reflect differences in habitual loads during growth and past ability to adapt, while response to loading reflects current individual physiologic capacity to adapt.

category: physiology


title: Informative neural representations of unseen objects during higher-order processing in human brains and deep artificial networks

abstract: A framework to pinpoint the scope of unconscious processing is critical to improve our models of visual consciousness. Previous research observed brain signatures of unconscious processing in visual cortex but these were not reliably identified. Further, whether unconscious content is represented in high-level stages of the ventral visual stream and linked parieto-frontal areas remains unknown. Using a within-subject, high-precision fMRI approach, we show that unconscious contents can be decoded from multivoxel patterns that are highly distributed alongside the ventral visual pathway and also involving parieto-frontal substrates. Classifiers trained with multivoxel patterns of conscious items generalised to predict the unconscious counterparts, indicating that their neural representations overlap. These findings suggest revisions to models of consciousness such as the neuronal global workspace. We then provide a computational simulation of visual processing/representation without perceptual sensitivity by using deep neural networks performing a similar visual task. The work provides a framework for pinpointing the representation of unconscious knowledge across different task domains.


title: Informative neural representations of unseen contents during higher-order processing in human brains and deep artificial networks


title: In vivo multi-parametric manganese-enhanced MRI for detecting senile plaques in rodent models of Alzheimer's disease

abstract:
Senile plaques are a hallmark of Alzheimers disease (AD) that develop in its earliest stages. Thus, non-invasive detection of these plaques would be invaluable for diagnosis and the development and monitoring of treatments, but this remains a challenge due to their small size. Here, we investigated the utility of manganese-enhanced MRI (MEMRI) for visualizing plaques in transgenic rodent models of AD across two species: 5xFAD mice and TgF344-AD rats.

Fourteen mice (eight transgenic, six wild-type) and eight rats (four transgenic, four wild-type) were given subcutaneous injections of MnCl2 and imaged in vivo using a 9.4T Bruker scanner. Susceptibility-weighted images, transverse relaxation rate (R2*) maps, and quantitative susceptibility maps were derived from high-resolution 3D multi-gradient-echo (MGE) data to directly visualize plaques. Longitudinal relaxation rate (R1) maps were derived from MP2RAGE data to measure regional manganese uptake. After scanning, the brains were processed for histology and stained for beta-amyloid (4G8 antibody), iron (Perls), and calcium/manganese (Alizarin Red).

MnCl2 improved signal-to-noise ratio (1.55{+/-}0.39-fold increase in MGE images) as expected, although this was not necessary for detection of plaques in the high-resolution images. Plaques were visible in susceptibility-weighted images, R2* maps, and quantitative susceptibility maps, with increased R2* and more positive magnetic susceptibility compared to surrounding tissue.

In the 5xFAD mice, most MR-visible plaques were in the hippocampus, though histology confirmed plaques in the cortex and thalamus as well. In the TgF344-AD rats, many more plaques were MR-visible throughout the hippocampus and cortex. Beta-amyloid and iron staining indicate that, in both models, MR visibility was driven by plaque size and iron load.

Voxel-wise comparison of R1 maps revealed increased manganese uptake in brain regions of high plaque burden in transgenic animals compared to their wild-type littermates. Interestingly, in contrast to plaque visibility in the high-resolution images, the increased manganese uptake was limited to the rhinencephalon in the TgF344-AD rats (family-wise error (FWE)-corrected p < 0.05) while it was most significantly increased in the cortex of the 5xFAD mice (FWE-corrected p < 0.3). Alizarin Red staining suggests that manganese bound to plaques in 5xFAD mice but not in TgF344-AD rats.

Multi-parametric MEMRI is a simple, viable method for detecting senile plaques in rodent models of AD. Manganese-induced signal enhancement can enable higher-resolution imaging, which is key to visualizing these small amyloid deposits. We also present in vivo evidence of manganese as a potential targeted contrast agent for imaging plaques in the 5xFAD model of AD.

HighlightsO_LIThis is the first study to use manganese-enhanced MRI (MEMRI) for direct visualization of senile plaques in rodent models of Alzheimers disease, in vivo.
C_LIO_LIManganese enhancement is not necessary to detect plaques but improves image contrast and signal-to-noise ratio.
C_LIO_LIManganese binds to plaques in 5xFAD mice but not in TgF344-AD rats, demonstrating potential as a targeted contrast agent for imaging plaques in certain models of AD.
C_LI


title: Intracranial brain stimulation modulates fMRI-based network switching

abstract: The extent to which functional MRI (fMRI) reflects direct neuronal changes remains unknown. Using 160 simultaneous electrical stimulation (es-fMRI) and intracranial brain stimulation recordings acquired in 26 individuals with epilepsy (with varying electrode locations), we tested whether brain networks dynamically change during intracranial brain stimulation, aiming to establish whether switching between brain networks is reduced after intracranial brain stimulation. As the brain spontaneously switches between a repertoire of intrinsic functional network configurations and the rate of switching is typically increased in brain disorders, we hypothesised that intracranial stimulation would reduce the brains switching rate, thus potentially normalising aberrant brain network dynamics. To test this hypothesis, we quantified the rate that brain regions changed networks over time in response to brain stimulation, using network switching applied to multilayer modularity analysis of time-resolved es-fMRI connectivity. Network switching and synchrony was decreased after the first brain stimulation followed by a more consistent pattern of network switching over time. This change was commonly observed in cortical networks and adjacent to the electrode targets. Our results suggest that neuronal perturbation is likely to modulate large-scale brain networks, and multilayer network modelling may be used to inform the clinical efficacy of brain stimulation in epilepsy.


title: Uneven declines between corals and cryptobenthic fish symbionts from multiple disturbances

abstract: With the onset and increasing frequency of multiple disturbances, the recovery potential of critical ecosystem-building species and their mutual symbionts is threatened. Similar effects to both hosts and their symbionts following disturbances have been assumed. However, we report unequal declines between hosts and symbionts throughout multiple climate-driven disturbances in reef-building Acropora corals and cryptobenthic coral-dwelling Gobiodon gobies. Communities were surveyed before and after consecutive cyclones (2014, 2015) and heatwaves (2016, 2017). After cyclones, coral size and goby group size decreased similarly by 28-30%. After heatwave-induced bleaching, coral size decreased substantially (47%) and the few gobies recorded mostly inhabited corals singly. Despite several coral species still occurring after bleaching, all goby species declined, leaving 78% of corals uninhabited. These findings suggest that gobies, which are important mutual symbionts for corals, are unable to cope with consecutive disturbances. This disproportionate decline could lead to ecosystem-level disruptions through loss of key symbiotic services to corals.


title: Establishment of a closed artificial ecosystem to ensure human long-term survival on the moon

abstract: Bioregenerative life support system (BLSS) is a critical technology maintaining long-term human survival on the Moon or other extraterrestrial bodies. In the current study, we carried out a 370-day integrated high-closure experiment ("Lunar Palace 365" experiment) on the Earth in an upgraded ground-based BLSS experimental facility called "Lunar Palace 1". This experiment was designed to develop techniques to run and adjust system stability under long-term operation and crew shift change conditions. Eight volunteering crew members were divided into two groups with three time phases: Group I stayed in the cabin for the initial 60-day phase; Group II inhabited the cabin instead for a record-breaking duration of 200 days as the second phase; Group I re-entered the cabin, replaced Group II and stayed for the last 105 days. Our results demonstrated the BLSS had excellent stability with a material closure degree of 98.2%. Levels of O2, CO2 and trace harmful gases were well controlled within ranges optimal for crew health and plant production. The system exhibited a strong robustness and could quickly minimize effects of disturbances through self-feedback adjustments. The efficiency of plant production completely met the crews need of plant-based food. The purification efficiency of domestic and sanitary wastewater was up to irrigation standards, and the recovery rate of urine and solid waste achieved 99.7% and 67%, respectively. These results are valuable for further optimization of the BLSS in a lunar base and computer simulations of similar systems.


title: Hybridization enables the fixation of selfish queen genotypes in eusocial colonies

abstract: A eusocial colony typically consists of two main castes: queens that reproduce and sterile workers that help them. This division of labour however is vulnerable to genetic elements that favour the development of their carriers into queens. Several factors, such as intra-colonial relatedness, can modulate the spread of such caste-biasing genotypes. Here we investigate the effects of a notable yet understudied ecological setting: where larvae produced by hybridization develop into sterile workers. Using mathematical modelling, we show that the coevolution of hybridization with caste determination readily triggers an evolutionary arms race between non-hybrid larvae that increasingly develop into queens, and queens that increasingly hybridize to produce workers. Even where hybridization reduces worker function and colony fitness, this race can lead to the loss of developmental plasticity and to genetically hard-wired caste determination. Overall, our results may help understand the repeated evolution towards remarkable reproductive systems (e.g. social hybridogenesis) observed in many ant species.


title: Using a biologically mimicking climbing robot to explore the performance landscape of climbing in lizards

abstract:
The life and death of an organism often depends on its ability to perform well at some ecologically relevant task. Yet despite this significance we have little idea how well species are optimised for competing locomotor tasks. Most scientists generally accept that the ability for natural systems to become optimised for a specific task is limited by structural, historic or functional constraints. Climbing lizards provide a good example of constraint where climbing ability requires the optimization of conflicting tasks such as speed, stability, or efficiency. Here we reconstruct multiple performance landscapes of climbing locomotion using a 10-DOF robot based upon the lizard bauplan, including an actuated spine, shoulders, and feet, the latter which interlock with the surface via claws. This design allows us to independently vary speed, foot angles, and range of motion, while simultaneously collecting data on climbed distance, stability and efficiency. We first demonstrate a trade-off between speed and stability with high speeds resulting in decreased stability and low speeds an increased cost of transport. By varying foot orientation of fore and hindfeet independently, we found geckos converge on a narrow optimum for both speed and stability, but avoid a secondary wider optimum highlighting a possible constraint. Modifying the spine and limb range of movement revealed a gradient in performance. Evolutionary modifications in movement among extant species appear to follow this gradient towards areas which promote speed and efficiency. This approach can give us a better understanding about locomotor optimization, and provide inspiration for industrial and search-and-rescue robots.

Significance StatementClimbing requires the optimization of conflicting tasks such as speed, stability, or efficiency, but understanding the relative importance of these competing performance traits is difficult.

We used a highly modular bio-inspired climbing robot to reconstruct performance landscapes for climbing lizards. We then compared the performance of extant species onto these and show strong congruence with lizard phenotypes and robotic optima.

Using this method we can show why certain phenotypes are not present among extant species, illustrating why these would be potentially mal-adaptive.

These principles may be useful to compare with relative rates of evolution along differing evolutionary histories. It also highlights the importance of biological inspiration towards the optimization of industrial climbing robots, which like lizards, must negotiate complex environments.


title: The influence of positive emotion and negative emotion on false memory based on EEG signals analysis

abstract: Analyzing the influence of emotion on false memory through electroencephalogram is helpful to further explore the cognition function of brain. In this study, we improved the Deese-Roediger-McDermott paradigm experiment to study the false memory. The memory materials are combined with mixed emotions, which are closer to real life. Twenty-eight participants were randomly divided into positive group and negative group. We used music to induce the participants in the positive group and the negative group to generate corresponding emotions. Finally, we analyzed the difference between the positive group and the negative group from the behavior data, source location and cortex functional network of event related potential. The results of behavioral data analysis show that the false memory rate of positive group (85%) is significantly higher than that of negative group (72%). The results of source localization show that the brain of the positive group is more active than that of the negative group, and the difference of brain activation location between the two groups is mainly manifested in the BA24 and BA45 brain regions. The results of cortex functional network show that the node degree, clustering coefficient, global efficiency and small-world property of the positive group are significantly higher than those of the negative group. It can be found from the three aspects that participants emotional state and brains understanding of semantic are the main reasons for the difference in the incidence of false memory between the two groups.


title: Biodegradable and Biocompatible Graphene-based Scaffolds for Functional Neural Tissue Engineering: A Strategy Approach Using Dental Pulp Stem Cells and Biomaterials

abstract: Neural tissue engineering aims to restore function of nervous system tissues using biocompatible cell-seeded scaffolds. Graphene-based scaffolds combined with stem cells deserve special attention to enhance tissue regeneration in a controlled manner. However, it is believed that minor changes in scaffold biomaterial com-position, internal porous structure, and physicochemical properties can impact cellular growth and adhesion. The current work aims to investigate in vitro biological effects of 3D graphene oxide (GO)/sodium alginate (GOSA) and reduced GOSA (RGOSA) scaffolds on dental pulp stem cells (DPSCs) in terms of cell viability and cytotoxicity. Herein, the effects of the 3D scaffolds, coating conditions, and serum supplementation on DPSCs functions are explored extensively. Biodegradation analysis revealed that addition of GO enhanced the degradation rate of composite scaffolds. Compared to the 2D surface, the cell viability of 3D scaffolds was higher (p <0.0001), highlighting the optimal initial cell adhesion to the scaffold surface and cell migration through pores. Moreover, the cytotoxicity study indicated that the incorporation of graphene supported higher DPSCs viability. It is also shown that when the mean pore size of scaffold increases, DPSCs activity decreases. In terms of coating conditions, poly-l-lysine (PLL) was the most robust coating reagent that improved cell-scaffold adherence and DPSCs metabolism activity. The cytotoxicity of GO-based scaffolds showed that DPSCs can be seeded in serum-free media without cytotoxic effects. This is critical for human translation as cellular transplants are typically serum-free. These findings suggest that proposed 3D GO-based scaffolds have favourable effects on the biological responses of DPSCs.

category: bioengineering


title: Super-Resolution Cryo-EM Maps With 3D Deep Generative Networks

abstract: An increasing number of biological macromolecules have been solved with cryo-electron microscopy (cryo-EM). Over the past few years, the resolutions of density maps determined by cryo-EM have largely improved in general. However, there are still many cases where the resolution is not high enough to model molecular structures with standard computational tools. If the resolution obtained is near the empirical border line (3-4 [A]), a small improvement of resolution will significantly facilitate structure modeling. Here, we report SuperEM, a novel deep learning-based method that uses a three-dimensional generative adversarial network for generating an improved-resolution EM map from an experimental EM map. SuperEM is designed to work with EM maps in the resolution range of 3 [A] to 6 [A] and has shown an average resolution improvement of 1.0 [A] on a test dataset of 36 experimental maps. The generated super-resolution maps are shown to result in better structure modelling of proteins.


title: Multidimensional Boolean Patterns in Multi-omics Data

abstract:
MotivationVirtually all biological systems are governed by a set of complex relations between their components. Identification of relations within biological systems involves a rigorous search for patterns among variables/parameters. Two-dimensional (involving two variables) patterns are identified using correlation, covariation, and mutual information approaches. However, these approaches are not suited to identify more complicated multidimensional relations, which simultaneously include 3, 4, and more variables.

ResultsWe present a novel pattern-specific method to quantify the strength and estimate the statistical significance of multidimensional Boolean patterns in multiomics data. In contrast with dimensionality reduction and AI solutions, patterns identified by the proposed approach may provide a better background for meaningful mechanistic interpretation of the biological processes. Our preliminary analysis suggests that multidimensional patterns may dominate the landscape of multi-omics data, which is not surprising because complex interactions between components of biological systems are unlikely to be reduced to simple pairwise interactions.


title: An optimized FM-index library for nucleotide and amino acid search

abstract: Pattern matching is a key step in a variety of biological sequence analysis pipelines. The FM-index is a compressed data structure for pattern matching, with search run time that is independent of the length of the database text. We present AvxWindowedFMindex (AWFM-index), an open-source, thread-parallel FM-index library written in C that is optimized for indexing nucleotide and amino acid sequences. AWFM-index is easy to incorporate into bioinformatics software and is able to perform exact match count and locate queries ~2-4x faster than SeqAn3s FM-index implementation for nucleotide search, and ~2-6x faster for amino acid search in a single-threaded context. This performance is due to (i) a new approach to storing FM-index data in a strided bit-vector format that enables extremely efficient computation of the FM-index occurrence function via AVX2 bitwise instructions, and (ii) inclusion of a cache-efficient lookup table for partial k-mer searches. AWFM-index also trivially parallelizes to multiple threads with good scaling, and enables efficient on-disk storage of the memory-intensive suffix array. The open-source library is available for download at https://github.com/TravisWheelerLab/AvxWindowFmIndex.


title: GN1, Glucosamine Analog, attenuate the neuronal hyperactivity of c-fos gene in Adjuvant-Induced Arthritis in Rats

abstract: The anti-inflammatory and anti-arthritic activity of GN1 (6-hydroxymethyl-3-(1-methylene-allylamino)-tetrahydro-pyran-2,4,5-triol), an analog of glucoseamine was investigated in the adjuvant induced arthritis (AIA) in rats. It was observed that the highly increased hind paw swelling was significantly reduced (p <0.001) with no noticeable retardation of body weight in the GN1 treated arthritic rats as compared to arthritic control rats. The histopathological analysis of isolated rat joints by scaling system classification of different stages appear in arthritic inflammatory lesion development, disclosed the suppressive effect in the inflammation processing in the GN1 treated animals. Different predictive markers such as TNF-, nitric oxide IL-1{beta}, peroxide and glutathione were also determined in the serum samples of the treated and non-treated arthritic rats in order to measure the intensity of inflammation. Our results show that GN-1 treatment in arthritic groups demonstrated marked reduction in c-fos expression. Results also revealed that GN1 significantly reduces the peroxide production (p < 0.002) and both the pro-inflammatory cytokines TNF- (p < 0.015) and IL-1{beta} (p < 0.001) in the arthritic rats receiving this treatment. Although, nitric oxide formation was found to be less than the arthritic control, but this reduction was not significant. The level of glutathione was significantly increased in GN1 treated arthritic rats (p < 0.037). These observations suggest that GN1 may help in reducing the inflammation and have anti-arthritic properties.


title: Proximal and Distal Nephron-specific Adaptation to Furosemide

abstract: Furosemide, a widely prescribed diuretic for edema-forming states, inhibits sodium reabsorption in the thick ascending limb of the nephron. Tubular adaptation to diuretics has been observed, but the range of mechanisms along the nephron has not been fully explored. Using morphometry, we show that furosemide induces renal tubular epithelial hyperplasia selectively in distal nephron segments. By comparison, we find progressive cellular hypertrophy in proximal and distal nephron segments. We next utilize single cell RNA sequencing of vehicle- and furosemide-treated mice to define potential mechanisms of diuretic resistance. Consistent with distal tubular cell hyperplasia, we detect a net increase in DCT cell number and Birc5, an anti-apoptotic and pro-growth gene, in a subset of DCT cells, as the most prominently up-regulated gene across the nephron. We also map a gradient of cell-specific transcriptional changes congruent with enhanced distal sodium transport. Furosemide stimulates expression of the mitogen IGF-1. Thus, we developed a mouse model of inducible deletion of renal tubular IGF-1 receptor and show reduced kidney growth and proximal, but not distal, tubular hypertrophy by furosemide. Moreover, genes that promote enhanced bioavailability of IGF-1 including Igfbp1 and Igfbp5 are significantly and differentially expressed in proximal tubular segments and correspond to IGF-1R-dependent hypertrophy. In contrast, downstream PI3-kinase signaling genes including Pdk1, Akt1, Foxo3, FKBP4, Eif2BP4, and Spp1 are significantly and differentially expressed in distal nephron segments and correspond to IGF-1R-independent hypertrophy. These findings highlight novel mechanisms of tubular remodeling and diuretic resistance, provide a repository of transcriptional responses to a common drug, and expand the implications of long-term loop diuretic use for human disease.


title: A convergent structure-function substrate of cognitive imbalances in autism

abstract: Autism is a common neurodevelopmental condition characterized by substantial phenotypic heterogeneity, which hinders diagnosis, research, and intervention. A leading example can be found in marked imbalances in language and perceptual skills, where deficits in one domain often co-exist with normal or even superior performance in the other domain. The current work capitalized on multiple data analytics including data-driven subtyping and dimensional approaches to quantify cognitive imbalances in multi-site datasets of individuals diagnosed with autism spectrum disorder (ASD) and neurotypical controls, and assessed structural and functional brain network substrates. Studying cognitive dimensions as well as multimodal neuroimaging signatures in 155 ASD and 151 neurotypical individuals, we observed robust evidence for a structure-function substrate of cognitive imbalances in ASD. Specifically, ASD presented with marked imbalances in cognitive profiles relative to neurotypical controls, characterized by verbal to non-verbal intelligence discrepancy. Different analytical approaches including subtyping and dimensional regression methods converged in showing that these imbalances were reflected in atypical cortical thickening and functional integration of language networks, alongside with sensory and higher cognitive networks. Phenotypic findings could be replicated in an independent sample of 325 ASD and 569 neurotypical controls. Although verbal and non-verbal intelligence are currently considered as specifiers unrelated to the categorical diagnosis of autism, our results show that intelligence disparities are accentuated in ASD and relate to a consistent structure-function substrate affecting multiple brain networks. Our findings motivate the incorporation of cognitive imbalances in future autism research, which may help to parse the phenotypic heterogeneity of autism and potentially inform intervention-oriented subtyping.


title: Layer 6A pyramidal cells subtypes form synaptic microcircuits with distinct functional and structural properties

abstract: Neocortical layer 6 plays a crucial role in sensorimotor coordination and integration through functionally segregated circuits linking intracortical and subcortical areas. However, because of the high neuronal heterogeneity and sparse intralaminar connectivity data on the cell-type specific synaptic microcircuits in layer 6 remain few and far between. To address this issue, whole-cell recordings combined with morphological reconstructions have been used to identify morpho-electric types of layer 6A pyramidal cells (PCs) in rat barrel cortex. Cortico-thalamic (CT), cortico-cortical (CC) and cortico-claustral (CCla) pyramidal cells have been distinguished based on to their distinct dendritic and axonal morphologies as well as their different electrophysiological properties. Here we demonstrate that these three types of layer 6A pyramidal cells innervate neighboring excitatory neurons with distinct synaptic properties: CT PCs establish weak facilitating synapses to other L6A PCs; CC PCs form synapses of moderate efficacy; while synapses made by putative CCla PCs display the highest release probability and a marked short-term depression. Furthermore, for excitatory-inhibitory synaptic connections in layer 6 we were able to show that both the presynaptic PC type and the postsynaptic interneuron type govern the dynamic properties of the of the respective synaptic connections. We have identified a functional division of local layer 6A excitatory microcircuits which may be responsible of the differential temporal engagement of layer 6 feed-forward and feedback networks. Our results provides a basis for further investigations on the long-range cortico-cortical, cortico-thalamic and cortico-claustral pathways.


title: FOXO1 and FOXO3 cooperatively regulate innate lymphoid cell development

abstract: The natural killer (NK) and non-cytotoxic innate lymphoid cells (ILC) lineages play vital role in the regulation of the immune system. Yet understanding of mechanisms controlling NK/ILC development remains incomplete. The evolutionary conserved FOXO family of forkhead transcription factors are critical regulators of cellular processes. We found that the loss of FOXO1 and FOXO3 together caused impaired activation of the NK gene expression program and reduced ETS binding already at the common lymphoid progenitor (CLP) level and a block at the ILC progenitor (ILCP) to NK progenitor transition. FOXO controlled NK cell maturation in organ specific manner and their ability to respond to IL-15. At the ILCP level, disruption of the ILC lineage specific gene programs was associated with broad perturbation of the generation of the non-cytotoxic ILC subsets. We concluded that FOXO1 and FOXO3 cooperatively regulate ILC lineage specification at the progenitor level as well as the generation of mature ILCs.


title: The Amot/Integrin protein complex transmits mechanical forces required for vascular expansion

abstract: Vascular development is a complex multistep process involving the coordination of cellular functions such as migration, proliferation and differentiation. Understanding the underlying mechanisms of these processes is of importance due to involvement of vessel expansion in various pathologies. How mechanical forces generated by cells and transmission of these physical forces control vascular development is poorly understood. Using an endothelial-specific genetic model in mice, we show that deletion of the scaffold protein, Angiomotin (Amot), inhibits migration and expansion of physical and pathological vascular network. We further show that Amot is required for tip cell migration and the extension of cellular filopodia. Exploiting in vivo and in vitro molecular approaches, we show that Amot binds talin and is essential for relaying forces between fibronectin and the cytoskeleton. Finally, we provide evidence that Amot is a novel component of the endothelial integrin adhesome and propose that Amot integrates spatial cues from the extra-cellular matrix in order to form a functional vascular network.


title: Chemical synthesis and purification of a non-hydrolyzable CTP analog CTPγS

abstract: Slowly- or non-hydrolyzable analogs of ATP and GTP, for example adenosine 5-({gamma}-thio)triphosphate (ATP{gamma}S) and guanosine 5-({gamma}-thio)triphosphate (GTP{gamma}S), have been widely employed to probe the roles of ATP and GTP in biosystems, and these compounds are readily available from commercial sources. In contrast, cytosine 5-({gamma}-thio)triphosphate (CTP{gamma}S) is not widely available commercially. The recent discovery of ParB as the founding member of a new class of CTPase enzyme (Osorio-Valeriano et al., 2019; Soh et al., 2019) and the possibility of multiple other undiscovered CTPases necessitate the use of the CTP{gamma}S to elucidate the roles of CTP hydrolysis in these systems. Here, we adapt a procedure for the synthesis of modified phosphoanhydrides (Hofer et al., 2015) to chemically synthesize and purify a milligram-scale of CTP{gamma}S.

category: biochemistry


title: Improving integrative 3D modeling into low- to medium- resolution EM structures with evolutionary couplings

abstract: Electron microscopy (EM) continues to provide near-atomic resolution structures for well-behaved proteins and protein complexes. Unfortunately, structures of some complexes are limited to low- to medium-resolution due to biochemical or conformational heterogeneity. Thus, the application of unbiased systematic methods for fitting individual structures into EM maps is important. A method that employs co-evolutionary information obtained solely from sequence data could prove invaluable for quick, confident localization of subunits within these structures. Here, we incorporate the co-evolution of intermolecular amino acids as a new type of distance restraint in the Integrative Modeling Platform (IMP) in order to build three-dimensional models of atomic structures into EM maps ranging from 10-14 [A] in resolution. We validate this method using four complexes of known structure, where we highlight the conservation of intermolecular couplings despite dynamic conformational changes using the BAM complex. Finally, we use this method to assemble the subunits of the bacterial holo-translocon into a model that agrees with previous biochemical data. The use of evolutionary couplings in integrative modeling improves systematic, unbiased fitting of atomic models into medium- to low-resolution EM maps, providing additional information to integrative models lacking in spatial data.


title: Divalent ligand-monovalent molecule binding

abstract: Simultaneous binding of divalent ligands to two identical molecules is a widespread phenomenon in biology and chemistry. Here, we describe this binding event as a divalent ligand AA that can bind to two identical monovalent molecules B to form the complex AA {middle dot} B2. Cases where the total concentration [AA]T is either much larger or much smaller than the total concentration [B]T have been studied earlier, but a description of intermediate concentrations is missing. In this paper, we describe the general case of any ratio of{xi} {equiv} [B]T /[AA]T. We show that the concentration of the intermediate complex AA {middle dot} B is governed by a cubic equation and discuss several scenarios in which this cubic equation simplifies. Our numerical results, which cover the entire range of 0 <{xi} < {infty}, are relevant to processes wherein the concentrations of free ligands and proteins both decrease upon binding. Such ligand and protein depletion is expected to be important in cellular contexts, e.g., in antigen detection and in coincidence detection of proteins or lipids.


title: Ciliary chemosensitivity is enhanced by cilium geometry and motility

abstract: Cilia are hairlike organelles involved in both sensory functions and motility. We discuss the question of whether the location of chemical receptors on cilia provides an advantage in terms of sensitivity. Using a simple advection-diffusion model, we compute the capture rates of diffusive molecules on a cilium. Because of its geometry, a non-motile cilium in a quiescent fluid has a capture rate equivalent to a circular absorbing region with ~ 4x its surface area. When the cilium is exposed to an external shear flow, the equivalent surface area increases to ~ 10x. Alternatively, if the cilium beats in a non-reciprocal way, its capture rate increases with the beating frequency to the power of 1/3. Altogether, our results show that the protruding geometry of a cilium could be one of the reasons why so many receptors are located on cilia. They also point to the advantage of combining motility with chemical reception.


title: N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

abstract: SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.


title: A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

abstract: The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of [~]80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 {degrees}C and were stable to long term storage of over 4 weeks at 37 {degrees}C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were [~] three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.


title: Immunogenicity and protective efficacy of a highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative


title: Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2

abstract:
The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.

IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.


title: The lethal triad: SARS-CoV-2 Spike, ACE2 and TMPRSS2. Mutations in host and pathogen may affect the course of pandemic.

abstract: Variants of SARS-CoV-2 have been identified rapidly after the beginning of pandemic. One of them, involving the spike protein and called D614G, represents a substantial percentage of currently isolated strains. While research on this variant was ongoing worldwide, on December 20th 2020 the European Centre for Disease Prevention and Control reported a Threat Assessment Brief describing the emergence of a new variant of SARS-CoV-2, named B.1.1.7, harboring multiple mutations mostly affecting the Spike protein. This viral variant has been recently associated with a rapid increase in COVID-19 cases in South East England, with alarming implications for future virus transmission rates. Specifically, of the nine amino acid replacements that characterize the Spike in the emerging variant, four are found in the region between the Fusion Peptide and the RBD domain (namely the already known D614G, together with A570D, P681H, T716I), and one, N501Y, is found in the Spike Receptor Binding Domain - Receptor Binding Motif (RBD-RBM). In this study, by using in silico biology, we provide evidence that these amino acid replacements have dramatic effects on the interactions between SARS-CoV-2 Spike and the host ACE2 receptor or TMPRSS2, the protease that induces the fusogenic activity of Spike. Mostly, we show that these effects are strongly dependent on ACE2 and TMPRSS2 polymorphism, suggesting that dynamics of pandemics are strongly influenced not only by virus variation but also by host genetic background.


title: Constitutive high expression of NOXA sensitizes human embryonic stem cells for rapid cell death

abstract:
Human embryonic stem (hES) cells are highly sensitive to apoptotic stimuli such as DNA damage, which allows for rapid elimination of mutated cells during development. However, the mechanisms that maintain hES cells in the primed apoptotic state are not completely known. Key activators of apoptosis, the BH3-only proteins, are present at low levels in most cell types. In contrast, hES cells have constitutive high levels of the BH3-only protein, NOXA. We examined the importance of NOXA for enabling apoptosis in hES cells. hES cells deleted for NOXA showed remarkable protection against multiple apoptotic stimuli. NOXA was constitutively localized to the mitochondria, where it interacted with MCL1. Strikingly, inhibition of MCL1 in NOXA knockout cells was sufficient to sensitize these cells to DNA damage, and subsequently, cell death. Our study demonstrates, an essential function of constitutive high levels of NOXA in hES cells is to effectively antagonize MCL1 to permit rapid apoptosis.

Significance statementHuman embryonic stem (hES) cells give rise to the entire organism, hence understanding how these cells regulate their survival and death is important. These cells undergo rapid death in response to DNA damage thereby removing mutated cells from the developing embryo. We focused on identifying the mechanism underlying the sensitivity of these cells to DNA damage. We discovered that the protein NOXA is essential for cell death in hES cells. Further, the crucial function of NOXA is to neutralize high levels of antiapototic protein, MCL1, thus enabling hES cells to respond rapidly to DNA damage.


title: Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

abstract: Promyelocytic leukemia (PML) proteins are involved in the pathogenesis of acute promyelocytic leukemia (APL). Trivalent arsenic (As3+) is known to cure APL by binding to cysteine residues of PML and enhance the degradation of PML-retinoic acid receptor  (RAR), a t(15;17) gene translocation product in APL cells, and restore PML-nuclear bodies (NBs). The size, number, and shape of PML-NBs vary among cell types and during cell division. However, topological changes of PML-NBs in As3+-exposed cells have not been well-documented. We report that As3+-induced solubility shift underlies rapid SUMOylation of PML and late aggregation of PML-NBs. Most PML-NBs were toroidal and irregular-shaped in GFPPML-transduced CHO-K1 and HEK293 cells, respectively. The annular PML-NBs appeared unstable and dissipated into small PML-NBs in HEK cells. Exposure to As3+ and antimony (Sb3+) greatly reduced the solubility of PML and enhanced SUMOylation within 2 h, and prolonged exposure resulted in PML-NB agglomeration. Exposure to bismuth (Bi3+), another Group 15 element, did not induce any of these changes. ML792, a SUMO activation inhibitor, reduced the number of PML-NBs and increased the size of the NBs, but had little effect on the As3+-induced solubility change of PML. The results show that SUMOylation regulates the dynamics of PML-NBs but does not contribute to the As3+-induced solubility change of PML.


title: Surveillance of genetic diversity and evolution in locally transmitted SARS-CoV-2 in Pakistan during the first wave of the COVID-19 pandemic

abstract: Surveillance of genetic diversity in the SARS-CoV-2 is extremely important to detect the emergence of more infectious and deadly strains of the virus. In this study, we monitored mutational events in the SARS-CoV-2 genome through whole genome sequencing. The samples (n=48) were collected from the hot spot regions of the metropolitan city Karachi, Pakistan during the four months (May 2020 to August 2020) of first wave of the COVID-19 pandemic. The data analysis highlighted 122 mutations, including 120 single nucleotide variations (SNV), and 2 deletions. Among the 122 mutations, there were 71 singletons, and 51 recurrent mutations. A total of 16 mutations, including 5 nonsynonymous mutations, were detected in spike protein. Notably, the spike protein missense mutation D614G was observed in 31 genomes. The phylogenetic analysis revealed majority of the genomes (36) classified as B lineage, where 2 genomes were from B.6 lineage, 5 genomes from B.1 ancestral lineage and remaining from B.1 sub-lineages. It was noteworthy that three clusters of B.1 sub-lineages were observed, including B.1.36 lineage (10 genomes), B.1.160 lineage (11 genomes), and B.1.255 lineage (5 genomes), which represent independent events of SARS-CoV-2 transmission within the city. The sub-lineage B.1.36 had higher representation from the Asian countries and the UK, B.1.160 correspond to the European countries with highest representation from the UK, Denmark, and lesser representation from India, Saudi Arabia, France and Switzerland, and the third sub-lineage (B.1.255) correspond to the USA. Collectively, our study provides meaningful insight into the evolution of SARS-CoV-2 lineages in spatio-temporal local transmission during the first wave of the pandemic.


title: An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+ conditions

abstract: The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+ against its transmembrane chemical potential gradient in low external K+ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+ conditions, K+ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition and the allosteric links between phosphorylation on the A domain and the inactivation of the pump remain unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC


title: Spatial drivers and pre-cancer populations collaborate with the microenvironment in untreated and chemo-resistant pancreatic cancer

abstract:
Pancreatic Ductal Adenocarcinoma (PDAC) is a lethal disease with limited treatment options and poor survival. We studied 73 samples from 21 patients (7 treatment-naive and 14 treated with neoadjuvant regimens), analyzing distinct spatial units and performing bulk proteogenomics, single cell sequencing, and cellular imaging. Spatial drivers, including mutant KRAS, SMAD4, and GNAQ, were associated with differential phosphosignaling and metabolic responses compared to wild type. Single cell subtyping discovered 12 of 21 tumors with mixed basal and classical features. Trefoil factor family members were upregulated in classical populations, while the basal populations showed enhanced expression of mesenchymal genes, including VIM and IGTB1. Acinar-ductal metaplasia (ADM) populations, present in 95% of patients, with 46% reduction of driver mutation fractions compared to tumor populations, exhibited suppressive and oncogenic features linked to morphologic states. We identified coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin receptor expression in tumor cells. Higher expression of angiogenic and stress response genes in dendritic cells compared to tumor cells suggests they have a pro-tumorigenic role in remodeling the microenvironment. Treated samples contain a three-fold enrichment of inflammatory CAFs when compared to untreated samples, while other CAF subtypes remain similar. A subset of tumor and/or ADM-specific biomarkers showed differential expression between treatment groups, and several known drug targets displayed potential cross-cell type reactivities. This resolution that spatially defined single cell omics provides reveals the diversity of tumor and microenvironment populations in PDAC. Such understanding may lead to more optimal treatment regimens for patients with this devastating disease.

HIGHLIGHTSO_LIAcinar-ductal metaplasia (ADM) cells represent a genetic and morphologic transition state between acinar and tumor cells.
C_LIO_LIInflammatory cancer associated fibroblasts (iCAFs) are a major component of the PDAC TME and are significantly higher in treated samples
C_LIO_LIReceptor-ligand analysis reveals tumor cell-TME interactions through NECTIN4-TIGIT
C_LIO_LITumor and ADM cell proteogenomics differ between treated and untreated samples, with unique and shared potential drug targets
C_LI

category: cancer biology


title: Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation

abstract: There is an urgent need to limit and stop the worldwide coronavirus disease 2019 (COVID-19) pandemic via quick development of efficient and safe vaccination methods. Plasmid DNA vaccines are one of the most remarkable vaccines that can be developed in a short term. pVAX1-SARS-CoV2-co, which is a plasmid DNA vaccine, was designed to express severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. The produced antibodies lead to Immunoreactions against S protein, anti-receptor-binding-domain, and neutralizing action of pVAX1-SARS-CoV2-co, as confirmed in a previous study. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine, a pyro-drive jet injector (PJI) was employed. PJI is an injection device that can adjust the injection pressure depending on various target tissues. Intradermally-adjusted PJI demonstrated that pVAX1-SARS-CoV2-co vaccine injection caused a strong production of anti-S protein antibodies, triggered immunoreactions and neutralizing actions against SARS-CoV-2. Moreover, a high dose of pVAX1-SARS-CoV2-co intradermal injection via PJI did not cause any serious disorders in the rat model. Finally, virus infection challenge in mice, confirmed that intradermally immunized (via PJI) mice were potently protected from COVID-19 infection. Thus, pVAX1-SARS-CoV2-co intradermal injection via PJI is a safe and promising vaccination method to overcome the COVID-19 pandemic.


title: A fragment-based protein interface design algorithm for symmetric assemblies

abstract: Theoretical and experimental advances in protein engineering have led to the creation of precisely defined, novel protein assemblies of great size and complexity, with diverse applications. One powerful approach involves designing a new attachment or binding interface between two simpler symmetric oligomeric protein components. The required methods of design, which present both similarities and key differences compared to problems in protein docking, remain challenging, and are not yet routine. With the aim of more fully enabling this emerging area of protein material engineering, we developed a computer program, Nanohedra, to introduce two key advances. First, we encoded in the program the construction rules (i.e. the search space parameters) that underlie all possible symmetric material constructions. Second, we developed algorithms for rapidly identifying favorable docking/interface arrangements based on tabulations of empirical patterns of known protein fragment-pair associations. As a result, the candidate poses that Nanohedra generates for subsequent amino acid interface design appear highly native-like (at the protein backbone level), while simultaneously conforming to the exacting requirements for symmetry-based assembly. A retrospective computational analysis of successful vs failed experimental studies supports the expectation that this should improve the success rate for this challenging area of protein engineering.

category: synthetic biology


title: Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue

abstract: A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies. Whilst untreated slices displayed no overt signs of pathology, exposure to tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau only arises at supra-physiological concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.


title: Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

abstract: Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.


title: Near-atomic resolution Cryo-EM structure of Mayaro virus identifies key structural determinants of alphavirus particle formation

abstract: Mayaro virus (MAYV) is an arthritis-inducing alphavirus circulating in the Americas, with potential to rapidly emerge in new geographical regions and populated environments. Intraparticle heterogeneity has typically limited atomic resolution structures of alphavirus virions, while imposing icosahedral symmetry in data processing prevents characterization of non-icosahedral features. Here, we report a near-atomic resolution cryo-EM structure of the MAYV E1-E2-E3-CP subunit by addressing deviations from icosahedral symmetry within each virus particle. We identified amino acid contacts at E1 protein interfaces forming the icosahedral lattice and investigated their effect on MAYV growth through site-directed mutagenesis. Further, mutation of a short stretch of conserved residues in E2 subdomain D, near an unidentified "pocket factor" including E2Y358, significantly reduced MAYV growth and provides strong evidence that this unknown factor influences assembly. Further, a symmetry-free reconstruction revealed the MAYV virion is not strictly icosahedral, suggesting defects in global symmetry may be a feature of the virus particle budding process. Our study provides insights into alphavirus assembly and suggests a common path in the formation of spherical, enveloped viruses, leading to particle imperfections.


title: Epidemiological and evolutionary consequences of CRISPR-Cas reactivity

abstract:
Adaptive immune systems face a control challenge: they should react with enough strength to clear an infection while avoiding to harm their organism. CRISPR-Cas systems are adaptive immune systems of prokaryotes that defend against fast evolving viruses. Here, we explore the CRISPR-Cas control challenge and look how its reactivity, i.e. its probability to acquire a new resistance, impacts the epidemiological outcome of a phage outbreak and the prokaryotes fitness. We show that in the absence of phage evolution, phage extinction is driven by the probability to acquire at least one resistance. However, when phage evolution is fast, phage extinction is characterised by an epidemiological critical threshold: any reactivity below this critical threshold leads to phage survival whereas any reactivity above it leads to phage extinction. We also show that in the absence of autoimmunity, high levels of reactivity evolve. However, when CRISPR-Cas systems are prone to autoimmune reactions, intermediate levels of reactivity are evolutionarily optimal. These results help explaining why natural CRISPR-Cas systems do not show high levels of reactivity.

Author summaryCRISPR-Cas systems are adaptive immune systems that use a complex 3-step molecular mechanism to defend prokaryotes against phages. Viral infections of populations defending with CRISPR-Cas can result in rapid phage extinction or in medium-term phage maintenance. What controls phage fate? Using mathematical modeling, we show that two parameters control this outcome: the phage escape rate and CRISPR-Cas reactivity (i.e. its probability of resistance acquisition upon infection). Furthermore, CRISPR-Cas reactivity impacts host fitness. From this, we derive that 1) CRISPR-Cas reactivity is a key predictor of the efficiency and of the cost of a CRISPR-Cas system, 2) there is an optimal reactivity balancing the cost of autoimmunity and immune efficiency and 3) high phage escape rate selects for higher CRISPR-Cas reactivities.


title: An enhancer sequence in the intrinsically disordered region of the essential cell division protein FtsZ promotes conformation-guided substrate processing by ClpXP in Escherichia coli

abstract: The essential bacterial division protein in Escherichia coli, FtsZ, assembles into the FtsZ-ring at midcell and recruits other proteins to the division site to promote septation. A region of the FtsZ amino acid sequence that links the conserved polymerization domain to a C-terminal protein interaction site was predicted to be intrinsically disordered and has been implicated in modulating spacing and architectural arrangements of FtsZ filaments. While the majority of cell division proteins that directly bind to FtsZ engage either the polymerization domain or the C-terminal interaction site, ClpX, the recognition and unfolding component of the bacterial ClpXP proteasome, has a secondary interaction with the predicted intrinsically disordered region (IDR) of FtsZ when FtsZ is polymerized. Here, we use NMR spectroscopy and reconstituted degradation reactions in vitro to demonstrate that this linker region is indeed disordered in solution and, further, that amino acids in the IDR of FtsZ enhance the degradation by conformationally-guided interactions.