title: CX3CL1 action on microglia protects from diet-induced obesity by restoring POMC neuronal excitability and melanocortin system activity impaired by high-fat diet feeding

abstract:
ObjectiveDiet-induced obesity (DIO) is associated with hypothalamic microglial activation and dysfunction of the melanocortin pathway, but the molecular mechanisms linking the two remain unclear. Previous studies have hypothesized that microglial inflammatory signaling is linked with impaired pro-opiomelanocortin (POMC) neuron function, but this mechanism has never been directly tested in vivo. We addressed this hypothesis using the specific microglial silencing molecule, CX3CL1 (fractalkine), to determine whether reducing hypothalamic microglial activation can restore POMC/melanocortin signaling in the brain to protect against DIO.

MethodsWe performed metabolic analyses in mice with targeted viral overexpression of CX3CL1 in the hypothalamus exposed to high fat diet (HFD). Electrophysiologic recording in hypothalamic slices from POMC-MAPT-GFP mice was used to determine the effects of HFD feeding and microglial silencing via minocycline or CX3CL1 on GFP-labeled POMC neurons. Finally, mice with hypothalamic overexpression of CX3CL1 received central treatment with the melanocortin receptor antagonist SHU-9119 to determine whether melanocortin signaling is required for the metabolic benefits of CX3CL1.

ResultsWe found that targeted expression of both soluble and membrane-bound forms of CX3CL1 in the mediobasal hypothalamus potently reduced weight gain and increased leptin sensitivity in animals exposed to high fat diet. The protective effect of CX3CL1 rescued diet-induced changes in POMC neuron excitability and required intact melanocortin receptor signaling in vivo.

ConclusionOur results provide the first evidence that HFD-induced POMC neuron dysfunction involves microglial activation. Furthermore, our study suggests that the anti-obesity action of CX3CL1 is mediated through the restoration of POMC neuron excitability and melanocortin signaling.


title: Correcting Cardiorespiratory Noise in Resting-state Functional MRI Data Acquired in Critically Ill Patients

abstract: Resting-state functional MRI (rs-fMRI) is being used to develop diagnostic, prognostic, and therapeutic biomarkers for critically ill patients with severe brain injuries. In studies of healthy volunteers and non-critically ill patients, prospective cardiorespiratory data are routinely collected to remove non-neuronal fluctuations in the rs-fMRI signal during analysis. However, the feasibility and utility of collecting cardiorespiratory data in critically ill patients on a clinical MRI scanner are unknown. We concurrently acquired rs-fMRI (TR=1250ms), cardiac and respiratory data in 23 critically ill patients with acute severe traumatic brain injury (TBI), and 12 healthy control subjects. We compared the functional connectivity results after denoising with cardiorespiratory data (i.e., RETROICOR) with the results obtained after standard bandpass filtering. Rs-fMRI data in 7 patients could not be analyzed due to imaging artifacts. In 6 of the remaining 16 patients (37.5%), cardiorespiratory data were either incomplete or corrupted. In both patients and control subjects, the functional connectivity corrected with RETROICOR did not significantly differ from that corrected with bandpass filtering of 0.008-0.125 Hz. Collectively, these findings suggest that there is a limited feasibility and utility to prospectively acquire high-quality cardiorespiratory data during rs-fMRI in critically ill patients with severe TBI for physiological correction.

category: neuroscience


title: Executive control by fronto-parietal activity explains counterintuitive decision behavior in complex value-based decision-making

abstract:
In real life, humans make decisions by taking into account multiple independent factors, such as delay and probability. Cognitive psychology suggests that cognitive control mechanisms play a key role when facing such complex task conditions. However, in value-based decision-making, it still remains unclear to what extent cognitive control mechanisms become essential when the task condition is complex. In this study, we investigated decision-making behaviors and underlying neural mechanisms using a multifactor gambling task where participants simultaneously considered probability and delay. Decision-making behavior in the multifactor task was modulated by both probability and delay. The behavioral effect of probability was stronger than delay, consistent with previous studies. Furthermore, in a subset of conditions that recruited fronto-parietal activations, reaction times were paradoxically elongated despite lower probabilistic uncertainty. Notably, such a reaction time elongation did not occur in control tasks involving single factors. Meta-analysis of brain activations suggested an association between the paradoxical increase of reaction time and strategy switching. Together, these results suggest a novel aspect of complex value-based decision-makings that is strongly influenced by fronto-parietal cognitive control.

Highlights* A value-based decision task with concurrent delay and probabilistic uncertainty
* Stronger behavioral effect of probability than delay
* Paradoxically long reaction time despite low probabilistic uncertainty
* The task activated fronto-parietal cognitive control network
* Reaction time elongation coincided with activation similar to strategy switching


title: Identifying Causal Subsequent Memory Effects

abstract: Over 40 years of accumulated research has detailed associations between neuroimaging signals measured during a memory encoding task and later memory performance, across a variety of brain regions, measurement tools, statistical approaches and behavioral tasks. But the interpretation of these Subsequent Memory Effects (SMEs) remains unclear: if the identified signals reflect cognitive and neural mechanisms of memory encoding then the underlying neural activity must be causally related to future memory. However, almost all previous SME analyses do not control for potential confounders of this causal interpretation, such as serial position and item effects. We collect a large fMRI dataset and use a novel experimental design and analysis approach that allows us to statistically adjust for all exogenous confounding variables. We find that, using standard approaches without adjustment, we replicate several univariate and multivariate subsequent memory effects and are able to predict memory performance across people. However, we are unable to identify any signal that reliably predicts subsequent memory after adjusting for confounding variables, bringing into doubt the causal status of these effects. We apply the same approach to subjects judgments of learning collected during an encoding period, and show that these behavioral measures of encoding quality do predict memory after adjustments, suggesting that it is possible to measure signals at the time of encoding that reflect causal mechanisms but that existing neuroimaging measures may not have the precision and specificity to do so.


title: Cerebral perfusion imaging: Hypoxia-induced deoxyhemoglobin or gadolinium?

abstract: Assessment of resting cerebrovascular perfusion measures (mean transit time, cerebral blood flow and cerebral blood volume) with magnetic resonance imaging currently requires the intravascular injection of the dynamic susceptibility contrast agent gadolinium. An initial comparison between hypoxia-induced deoxyhemoglobin and gadolinium was made for these measures in six healthy participants. A bolus of deoxyhemoglobin is generated in the lung via transient hypoxia induced by an available computer-controlled gas blender technology employing sequential gas delivery (RespirAct). We hypothesised and confirmed perfusion measures from both susceptibility contrast agents would yield similar spatial patterns of cerebrovascular perfusion measures. We conclude that hypoxia-induced deoxyhemoglobin, an endogenously, non-invasively generated, non-allergenic, non-toxic, recyclable, environmentally innocuous molecule, may be suitable to become the first new magnetic resonance imaging susceptibility contrast agent introduction since gadolinium.


title: A biologically inspired architecture with switching units can learn to generalize across backgrounds

abstract: Humans and other animals navigate different landscapes and environments with ease, a feat that requires the brains ability to rapidly and accurately adapt to different visual domains, generalizing across contexts/backgrounds. Despite recent progress in deep learning applied to classification and detection in the presence of multiple confounds including contextual ones [25, 30], there remain important challenges to address regarding how networks can perform context-dependent computations and how contextually-invariant visual concepts are formed. For instance, recent studies have shown artificial networks that repeatedly misclassified familiar objects set on new backgrounds, e.g. incorrectly labelling known animals when they appeared in a different setting [3]. Here, we show how a bio-inspired network motif can explicitly address this issue. We do this using a novel dataset which can be used as a benchmark for future studies probing invariance to backgrounds. The dataset consists of MNIST digits of varying transparency, set on one of two backgrounds with different statistics: a Gaussian noise or a more naturalistic background from the CIFAR-10 dataset. We use this dataset to learn digit classification when contexts are shown sequentially, and find that both shallow and deep networks have sharply decreased performance when returning to the first background after experience learning the second - the catastrophic forgetting phenomenon in continual learning. To overcome this, we propose an architecture with additional " switching" units that are activated in the presence of a new background. We find that the switching network can learn the new context even with very few switching units, while maintaining the performance in the previous context - but that they must be recurrently connected to network layers. When the task is difficult due to high transparency, the switching network trained on both contexts outperforms networks without switching trained on only one context. The switching mechanism leads to sparser activation patterns, and we provide intuition for why this helps to solve the task. We compare our architecture with other prominent learning methods, and find that elastic weight consolidation is not successful in our setting, while progressive nets are more complex but less effective. Our study therefore shows how a bio-inspired architectural motif can contribute to task generalization across context.


title: Parametric control of flexible timing through low-dimensional neural manifolds

abstract: Biological brains possess an unparalleled ability to generalize adaptive behavioral responses from only a few examples. How neural processes enable this capacity to extrapolate is a fundamental open question. A prominent but underexplored hypothesis suggests that generalization is facilitated by a low-dimensional organization of collective neural activity. Here we tested this hypothesis in the framework of flexible timing tasks where dynamics play a key role. Examining trained recurrent neural networks we found that confining the dynamics to a low-dimensional subspace allowed tonic inputs to parametrically control the overall input-output transform and enabled smooth extrapolation to inputs well beyond the training range. Reverse-engineering and theoretical analyses demonstrated that this parametric control of extrapolation relies on a mechanism where tonic inputs modulate the dynamics along non-linear manifolds in activity space while preserving their geometry. Comparisons with neural data from behaving monkeys confirmed the geometric and dynamical signatures of this mechanism.


title: Longitudinal immune cell profiling in early systemic lupus erythematosus

abstract:
ObjectiveTo investigate the immune cell profiling and their longitudinal changes in systemic lupus erythematosus (SLE).

MethodsWe employed mass cytometry with three different 38-39 marker panels (Immunophenotyping, T cell/monocyte, and B cell) in cryopreserved peripheral blood mononuclear cells (PBMCs) from nine patients with early SLE, 15 patients with established SLE, and 14 non-inflammatory controls. We used machine learning-driven clustering, FlowSOM (Flow Self-Organizing Maps) and dimensional reduction with tSNE (t-distributed Stochastic Neighbor Embedding) to identify unique cell populations in early and established SLE. For the nine early SLE patients, longitudinal mass cytometry analysis was applied to PBMCs at three time points (at enrollment, six months post-enrollment, and one year post-enrollment). Serum samples were also assayed for 65 cytokines by Luminex multiplex assay, and associations between cell types and cytokines/chemokines assessed.

ResultsT peripheral helper cells (Tph cells), T follicular helper cells (Tfh cells) and several Ki67+ proliferating subsets (ICOS+ Ki67+ CD8 T cells, Ki67+ regulatory T cells, CD19int Ki67hi plasmablasts, and Ki67hi PU.1hi monocytes) were increased in early SLE. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from early SLE patients revealed that Tfh cells and CXCL10 decreased at one year post-enrollment. CXCL13 correlated positively with several of the expanded cell populations in early SLE.

ConclusionsTwo major helper T cell subsets and unique Ki67+ proliferating immune cell subsets were expanded in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

category: immunology


title: Gene recruitments and dismissals in argonaut octopus genome provide insights to pelagic lifestyle adaptation and shell-like eggcase reacquisition

abstract: The paper nautilus, Argonauta argo, also known as the greater argonaut, is a species of octopods distinctly characterized by its pelagic lifestyle and by the presence of a spiral-shaped shell-like eggcase in females. The eggcase functions by protecting the eggs laid inside it, and by building and keeping air intakes for buoyancy. To reveal the genomic background of the species adaptation to pelagic lifestyle and the acquisition of its shell-like eggcase, we sequenced the draft genome sequence of the species. The genome size was 1.1 Gb, which is the smallest among the cephalopods known to date, with the top 215 scaffolds (average length 5,064,479 bp) covering 81% (1.09 Gb) of the total assembly. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified nearly intact HOX, Parahox, Wnt clusters and some gene clusters probably related to the pelagic lifestyle, such as reflectin, tyrosinase, and opsin. For example, opsin might have undergone an extensive duplication in order to adapt to the pelagic lifestyle, as opposed to other octopuses, which are mostly the benthic. Our gene models also discovered several genes homologous to those related to calcified shell formation in Conchiferan Mollusks, such as Pif-like, SOD, and TRX. Interestingly, comparative genomics analysis revealed that the homologous genes for such genes were also found in the genome of the octopus, which does not have a shell, as well as the basal cephalopods Nautilus. Therefore, the draft genome sequence of A. argo we presented here had not only helped us to gain further insights into the genetic background of the dynamic recruitment and dismissal of genes for the formation of an important, converging extended phenotypic structure such as the shell and the shell-like eggcase, but also the evolution of lifestyles in Cephalopods and the octopods, from benthic to pelagic.

category: genomics


title: Prospects of telomere-to-telomere assembly in barley: analysis of sequence gaps in the MorexV3 reference genome

abstract: The first gapless, telomere-to-telomere (T2T) sequence assemblies of plant chromosomes were reported recently. However, sequence assemblies of most plant genomes remain fragmented. Only recent breakthroughs in accurate long-read sequencing have made it possible to achieve highly contiguous sequence assemblies with a few tens of contigs per chromosome, i.e. a number small enough to allow for a systematic inquiry into the causes of the remaining sequence gaps and the approaches and resources needed to close them. Here, we analyze sequence gaps in the current reference genome sequence of barley cv. Morex (MorexV3). Optical map and sequence raw data, complemented by ChIP-seq data for centromeric histone variant CENH3, were used to estimate the abundance of centromeric, ribosomal DNA and subtelomeric repeats in the barley genome. These estimates were compared with copy numbers in the MorexV3 pseudomolecule sequence. We found that almost all centromeric sequences and 45S ribosomal DNA repeat arrays were absent from the MorexV3 pseudomolecules and that the majority of sequence gaps can be attributed to assembly breakdown in long stretches of satellite repeats. However, missing sequences cannot fully account for the difference between assembly size and flow cytometric genome size estimates. We discuss the prospects of gap closure with ultra-long sequence reads.


title: AWGAN: A Powerful Batch Correction Model for scRNA-seq Data

abstract: With the advancement of technology, we can generate and access large-scale, high dimensional and diverse genomics data, especially through single-cell RNA sequencing (scRNA-seq). However, integrative downstream analysis from multiple scRNA-seq datasets remains challenging due to batch effects. In this paper, we focus on scRNA-seq data integration and propose a new deep learning framework based on Wasserstein Generative Adversarial Network (WGAN) combined with an attention mechanism to reduce the differences among batches. We also discuss the limitations of the existing methods and demonstrate the advantages of our new model from both theoretical and practical aspects, advocating the use of deep learning in genomics research.

category: bioinformatics


title: hybpiper-rbgv and yang-and-smith-rbgv: Containerization and additional options for assembly and paralog detection in target enrichment data

abstract:
PREMISEThe HybPiper pipeline has become one of the most widely used tools for the assembly of target enrichment (sequence capture) data for phylogenomic analysis. Between the production of locus sequences and phylogenetic analysis, the identification of paralogs is a critical step ensuring accurate inference of evolutionary relationships. Algorithmic approaches using gene tree topologies for the inference of ortholog groups are computationally efficient and broadly applicable to non-model organisms, especially in the absence of a known species tree. Unfortunately, software compatibility issues, unfamiliarity with relevant programming languages, and the complexity involved in running numerous subsequent analysis steps continue to limit the broad uptake of these approaches and constrain their application in practice.

METHODS AND RESULTSWe updated the scripts constituting HybPiper and a pipeline for the inference of ortholog groups ("Yang and Smith") to provide novel options for the treatment of supercontigs, remove bugs, and seamlessly use the outputs of the former as inputs for the latter. The pipelines were containerised using Singularity and implemented via two Nextflow pipelines for easier deployment and to vastly reduce the number of commands required for their use. We tested the pipelines with several datasets, one of which is presented for demonstration.

CONCLUSIONShybpiper-rbgv and yang-and-smith-rbgv provide easy installation, user-friendly experience, and robust results to the phylogenetic community. They are presently used as the analysis pipeline of the Australian Angiosperm Tree of Life project. The pipelines are available at https://github.com/chrisjackson-pellicle.


title: scDeepHash: An automatic cell type annotation and cell retrieval method for large-scale scRNA-seq datasets using neural network-based hashing

abstract: Single-cell RNA-sequencing technologies measure transcriptomic expressions, which quantifies cell-to-cell heterogeneity at an unprecedented resolution. As these technologies become more readily available, the number of scRNA-seq datasets increases drastically. Prior works have demonstrated that bias-free, holistic single-cell profiling infrastructures are essential to the emerging automatic cell-type annotation methods. We propose scDeepHash, a scalable scRNA-seq analytic tool that employs content-based deep hashing to index single-cell gene expressions. scDeepHash allows for fast and accurate automated cell-type annotation and similar-cell retrieval. We also demonstrate the performance of scDeepHash by benchmarking it against current state-of-the-art methods across multiple public scRNA-seq datasets.


title: mTOR Inhibition Overcomes RSK3-mediated Resistance to BET Inhibitors in Small Cell Lung Cancer

abstract:
PurposeSCLC is a recalcitrant malignancy with limited treatment options. BET inhibitors have shown promising preclinical activity in SCLC, but their broad sensitivity spectrum limits their clinical prospects in this malignancy. Drug combination could be a solution.

Experimental designWe performed high-throughput drug combination screens in SCLC cell lines to identify potential therapeutics synergizing with BET inhibitors. Validation was performed in SCLC cell lines and patient-derived xenograft models. Genome-wide RNA sequencing of xenograft tumors was performed to determine the mechanism underlying the synergy of the drug combination.

ResultsInhibitors of the PI-3K-AKT-mTOR pathway were the top candidates from the screens. Among the therapeutics targeting this pathway, mTOR inhibitors showed the highest degree of synergy with BET inhibitors in vitro. Furthermore, the combination of these two classes of drugs showed superior antitumor efficacy and tolerability in vivo. Using both in vitro and in vivo SCLC models, we demonstrate that BET inhibitors activate the intrinsic apoptotic cascade, and mTOR inhibitors further enhance these apoptotic effects. Mechanistically, BET inhibitors activate the TSC2-mTOR-p70S6K1 signaling cascade by upregulating RSK3, an upstream kinase of TSC2. Activation of p70S6K1 leads to BAD phosphorylation and cell survival. mTOR inhibition blocks this survival signaling cascade and potentiates the antitumor effects of BET inhibitors.

ConclusionsOur results demonstrate that RSK3 upregulation is a novel resistance mechanism of BET inhibitors in SCLC, and mTOR inhibition can overcome this resistance and enhance apoptosis. These findings provide a rationale to evaluate the combination of mTOR and BET inhibitors in patients with SCLC.

category: cancer biology


title: Chromogranin A plays a crucial role in the age-related development of insulin resistance and hypertension

abstract: Aging is associated with the development of metabolic disorders, including insulin resistance and hypertension. Young mice that are negative for the neuroendocrine prohormone Chromogranin A (CgA knockout, CgA-KO) display two opposite aging phenotypes: hypertension but heightened insulin sensitivity. We determined these phenotypes in aging CgA mice. In comparison, aging wild-type (WT) mice gradually lost glucose tolerance and insulin sensitivity. Moreover, while aging WT mice had increased inflammation with higher plasma TNF-, IFN-{gamma} and CCL2 and increased mitochondrial fission, these phenotypes were the opposite in aging CgA-KO mice. CgA-KO mice also showed increased expression of mitochondrial and nuclear-encoded complex I genes, implying that they were healthier than WT mice. Most intriguingly, the hypertension in CgA-KO mice was spontaneously reversed with aging. Supplementation of CgA-KO mice with pancreastatin, a hyperglycemic peptide produced from CgA by proteolysis, increased both blood glucose levels and blood pressure, implicating hyperglycemia, and hypertension. We conclude that age-related insulin resistance and hypertension are caused by CgA.

category: physiology


title: Calcium and strontium contractile activation properties of single skinned skeletal muscle fibres from elderly women 66-90 years of age

abstract: The single skinned muscle fibre technique was used to investigate Ca2+- and Sr2+- activation properties of skeletal muscle fibres from elderly women (66-90 years). Muscle biopsies were obtained from the vastus lateralis muscle. Three populations of muscle fibres were identified according to their specific Sr2+- activation properties: slow-twitch (type I), fast-twitch (type II) and hybrid (type I/II) fibres. All three fibre types were sampled from the biopsies of 66 to 72 years old women, but the muscle biopsies of women older than 80 years yielded only slow-twitch (type I) fibres. The proportion of hybrid fibres in the vastus lateralis muscle of women of circa 70 years of age (24%) was several-fold greater than in the same muscle of adults (<10%), suggesting that muscle remodelling occurs around this age. There were no differences between the Ca2+- and Sr2+- activation properties of slow-twitch fibres from the two groups of elderly women, but there were differences compared with muscle fibres from adults with respect to sensitivity to Ca2+, steepness of the activation curves, and characteristics of the fibre-type dependent phenomenon of spontaneous force oscillations (SOMO) occurring at submaximal levels of activation. The maximal Ca2+ activated specific force from all the fibres collected from the seven old women use in the present study was significantly lower by 20% than in the same muscle of adults. Taken together these results show there are qualitative and quantitative changes in the activation properties of the contractile apparatus of muscle fibres from the vastus lateralis muscle of women with advancing age, and that these changes need to be considered when explaining observed changes in womens mobility with aging.


title: Bedtime to the brain: How infants sleep habits intertwine with sleep neurophysiology

abstract:
Adequate sleep is critical for development and facilitates the maturation of the neurophysiological circuitries at the basis of cognitive and behavioral function. Observational research has associated sleep problems in early life with worse later cognitive, psychosocial, and somatic health outcomes. Yet, the extent to which day-to-day sleep habits in early life relate to neurophysiology - acutely and long-term - remains to be explored. Here, we report that sleep habits in 32 healthy 6-month-olds assessed with actimetry are linked to fundamental aspects of their neurophysiology measured with high-density electroencephalography (hdEEG). Our study reveals four key findings: First, daytime sleep habits are linked to EEG slow wave activity (SWA). Second, habits of nighttime movement and awakenings from sleep are connected with spindle density. Third, habitual sleep timing is linked to neurophysiological connectivity quantified as Delta-coherence. And lastly, Delta-coherence at age 6 months predicts nighttime sleep duration at age 12 months. These novel findings widen our understanding that infants sleep habits are closely intertwined with three particular levels of neurophysiology: sleep pressure (determined by SWA), the maturation of the thalamocortical system (spindles), and the maturation of cortical connectivity (coherence). Our companion paper complements this insight in the perspective of later developmental outcomes: early thalamocortical connectivity (spindle density) at age 6 months predicts later behavioural status at 12 and 24 months. The crucial next step is to extend this concept to clinical groups to objectively characterize infants sleep habits "at risk" that foster later neurodevelopmental problems.

HighlightsO_LIInfants habitual sleep behavior (actimetry) is linked with their sleep neurophysiology (EEG)
C_LIO_LIHabits of daytime sleeping (naps) are related to slow wave activity
C_LIO_LIInfants movements and awakenings at nighttime are linked to their sleep spindles
C_LIO_LISleep timing (infants bedtimes) is associated with cortical connectivity in the EEG
C_LI


title: Individual differences in frontoparietal plasticity in humans

abstract: Neuroplasticity, defined as the brains potential to change in response to its environment, has been extensively studied at the cellular and molecular levels. Work in animal models suggests that stimulation to the ventral tegmental area (VTA) enhances plasticity, and that myelination constrains plasticity. Little is known, however, about whether proxy measures of these properties in the human brain are associated with learning. Here, we investigated the plasticity of the frontoparietal system by asking whether VTA resting-state functional connectivity and myelin map values (T1w/T2w ratios) predicted learning after short-term training on the adaptive n-back (n = 46, ages 18-25). We found that stronger baseline connectivity between VTA and lateral prefrontal cortex predicted greater improvements in accuracy. Lower myelin map values predicted improvements in response times, but not accuracy. Our findings suggest that proxy markers of neural plasticity can predict learning in humans.


title: Mice infected with Mycobacterium tuberculosis are resistant to secondary infection with SARS-CoV-2

abstract:
Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are the leading causes of death due to infectious disease. Although Mtb and CoV2 both cause serious and sometimes fatal respiratory infections, the effect of Mtb infection and its associated immune response on secondary infection with CoV2 is unknown. To address this question we applied two mouse models of COVID19, using mice which were chronically infected with Mtb. In both model systems, Mtb-infected mice were resistant to secondary CoV2 infection and its pathological consequences, and CoV2 infection did not affect Mtb burdens. Single cell RNA sequencing of coinfected and monoinfected lungs demonstrated the resistance of Mtb-infected mice is associated with expansion of T and B cell subsets upon viral challenge. Collectively, these data demonstrate that Mtb infection conditions the lung environment in a manner that is not conducive to CoV2 survival.

AUTHOR SUMMARYMycobacterium tuberculosis (Mtb) and SARS-CoV-2 (CoV2) are distinct organisms which both cause lung disease. We report the surprising observation that Mtb-infected mice are resistant to secondary infection with CoV2, with no impact on Mtb burden and resistance associating with lung T and B cell expansion.


title: Bone marrow mesenchymal stem cells stimulated by tumor necrosis factor-α can promote the repair of fatty liver cell oxidative stress injury and fatty liver ischemia-reperfusion injury

abstract: Mesenchymal stem cells (MSCs) have great prospects for the treatment of ischemia-reperfusion injury (IRI) after liver transplantation. At this stage, the main factor limiting MSCs in the treatment of fatty liver IRI of the donor liver is the residence time of stem cells at the site of inflammatory injury. This study investigated whether bone marrow mesenchymal stem cells (BMSCs) stimulated by tumor necrosis factor- (TNF-) can promote the repair of fatty liver cell oxidative stress injury and fatty liver IRI in rats. The results indicated the BMSCs treatment group stimulated by TNF- had lower indexes and significantly improved oxidative stress damage in vitro through Transwell chamber co-culture experiment, compared with the control group. In vivo, compared with the PBS group and the BMSCs group, the indexes of the BMSCs treatment group stimulated by TNF- were reduced, and the degree of tissue damage was significantly reduced. BMSCs can repair fatty liver cell oxidative stress injury and fatty liver IRI, however, BMSCs stimulated by TNF- can promote the repair of tissues and cells.

category: cell biology


title: Target of Rapamycin drives unequal responses to essential amino acid depletion in egg laying

abstract: Nutrition shapes a broad range of life history traits, ultimately impacting animal fitness. A key fitness-related trait, female fecundity is well known to change as a function of diet. In particular, the availability of dietary protein is one of the main drivers of egg production, and in the absence of essential amino acids egg laying declines. However, it is unclear whether all essential amino acids have the same impact on phenotypes like fecundity. Using a holidic diet, we fed adult female D. melanogaster diets that contain all necessary nutrients except one of the 10 essential amino acids and assessed the effects on egg production. For most essential amino acids, depleting a single amino acid induced as rapid a decline in egg production as when there were no amino acids in the diet. However, when either methionine or histidine were excluded from the diet, egg production declined more slowly. Next, we tested whether GCN2 and TOR were involved in this difference in response across amino acids. While mutations in GCN2 did not eliminate the differences in the rates of decline in egg laying among amino acid drop-out diets, we found that inhibiting TOR signalling caused egg laying to decline rapidly for all drop-out diets. TOR signalling does this by regulating the yolk-forming stages of egg chamber development. Our results suggest that amino acids differ in their ability to induce signalling via the TOR pathway. This is important because if phenotypes differ in sensitivity to individual amino acids, this generates the potential for mismatches between the output of a pathway and the animals true nutritional status.

category: developmental biology


title: Comparison of materials for rapid passive collection of environmental DNA

abstract:
Passive collection is an emerging sampling method for environmental DNA (eDNA) in aquatic systems. Passive eDNA collection is inexpensive, efficient, and requires minimal equipment, making it suited to high density sampling and remote deployment. Here, we compare the effectiveness of nine membrane materials for passively collecting fish eDNA from a 3 million litre marine mesocosm. We submerged materials (cellulose, cellulose with 1% and 3% chitosan, cellulose overlayed with electrospun nanofibers and 1% chitosan, cotton fibres, hemp fibres and sponge with either zeolite or active carbon) for intervals between five and 1080 minutes. We show that for most materials, with as little as five minutes submersion, mitochondrial fish eDNA measured with qPCR, and fish species richness measured with metabarcoding, was comparable to that collected by conventional filtering. Furthermore, PCR template DNA concentrations and species richness were generally not improved significantly by longer submersion. Species richness detected for all materials ranged between 11 to 37 species, with a median of 27, which was comparable to the range for filtered eDNA (19-32). Using scanning electron microscopy, we visualised biological matter adhered to the surface of materials, rather than entrapped, with images also revealing a diversity in size and structure of putative eDNA particles.

Environmental DNA can be collected rapidly from seawater with a passive approach and using a variety of materials. This will suit cost and time-sensitive biological surveys, and where access to equipment is limited.

category: ecology


title: Interpreting ruminant specific conserved non-coding elements by developmental gene regulatory network

abstract:
BackgroundBiologists long recognized that the genetic information encoded in DNA leads to trait innovation via gene regulatory network (GRN) in development.

ResultsHere, we generated paired expression and chromatin accessibility data during rumen and esophagus development in sheep and revealed 1,601 active ruminant-specific conserved non-coding elements (active-RSCNEs). To interpret the function of these active-RSCNEs, we developed a Conserved Non-coding Element interpretation method by gene Regulatory network (CNEReg) to define toolkit transcription factors (TTF) and model its regulation on rumen specific gene via batteries of active-RSCNEs during development. Our developmental GRN reveals 18 TTFs and 313 active-RSCNEs regulating the functional modules of the rumen and identifies OTX1, SOX21, HOXC8, SOX2, TP63, PPARG and 16 active-RSCNEs that functionally distinguish the rumen from the esophagus.

ConclusionsWe argue that CNEReg is an attractive systematic approach to integrate evo-devo concepts with omics data to understand how gene regulation evolves and shapes complex traits.


title: Design of D-amino acids SARS-CoV-2 Main protease inhibitors using the cationic peptide from rattlesnake venom as a scaffold

abstract: The C30 Endopeptidase (3C-like protease; 3CLpro) is essential for the life cycle of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) since it plays a pivotal role in viral replication and transcription and is hence a promising drug target. Molecules isolated from animals, insects, plants or microorganisms can serve as a scaffold for the design of novel biopharmaceutical products. Crotamine, a small cationic peptide from the venom of the rattlesnake Crotalus durissus terrificus has been the focus of many studies since it exhibits activities such as analgesic, in vitro antibacterial and hemolytic activities. The crotamine derivative L-peptides (L-CDP) that inhibit the 3CL protease in the low {micro}M range were examined since they are susceptible to proteolytic degradation; we explored the utility of their D-enantiomers form. Comparative uptake inhibition analysis showed D-CDP as a promising prototype for a D-peptide-based drug. We also found that the D-peptides can impair SARS-CoV-2 replication in vivo, probably targeting the viral protease 3CLpro.

category: biochemistry


title: Mitochondrial calcium uptake regulates tumour progression in embryonal rhabdomyosarcoma

abstract: Embryonal rhabdomyosarcoma (ERMS) is characterized by a failure of cells to complete skeletal muscle differentiation. Although ERMS cells are vulnerable to oxidative stress, the relevance of mitochondrial calcium homeostasis in oncogenesis is unclear. Here, we show that ERMS cell lines as well as primary tumours exhibit elevated expression of the Mitochondrial Calcium Uniporter (MCU). MCU knockdown resulted in impaired mitochondrial calcium uptake and a reduction in mitochondrial reactive oxygen species (mROS) levels. Phenotypically, MCU knockdown cells exhibited reduced cellular proliferation and motility, with an increased propensity to differentiate in vitro and in vivo. RNA-sequencing of MCU knockdown cells revealed a significant reduction in genes involved in TGF{beta} signalling that play prominent roles in oncogenesis and inhibition of myogenic differentiation. Interestingly, modulation of mROS production impacted TGF{beta} signalling. Our study elucidates mechanisms by which mitochondrial calcium dysregulation promotes tumour progression and suggests that targeting the MCU complex to restore mitochondrial calcium homeostasis could be a therapeutic avenue in ERMS.


title: Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations

abstract:
ObjectiveTo determine if mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis.

DesignThe ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis.

SettingThe vivarium of a research laboratory

SubjectsBalb/c mice

Interventions: Mice were challenged with an LD80 dose of either lipopolysaccharide (LPS / endotoxin), TNF, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis.

Measurement and Main ResultsEach group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs (1) were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time and dose dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animal if administered early and at a high dose.

ConclusionsOur conclusions are MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAbs efficacy is both time and dose dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens-of-thousands of lives annually and could result in improved antibiotic stewardship.


title: A virtual host model of Mycobacterium tuberculosis infection identifies early immune events as predictive of infection outcomes

abstract: Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb), is one of the worlds deadliest infectious diseases and remains a significant global health burden. TB disease and pathology can present clinically across a spectrum of outcomes, ranging from total sterilization of infection to active disease. Much remains unknown about the biology that drives an individual towards various clinical outcomes as it is challenging to experimentally address specific mechanisms driving clinical outcomes. Furthermore, it is unknown whether numbers of immune cells in the blood accurately reflect ongoing events during infection within human lungs. Herein, we utilize a systems biology approach by developing a whole-host model of the immune response to Mtb across multiple physiologic and time scales. This model, called HostSim, tracks events at the cellular, granuloma, organ, and host scale and represents the first whole-host, multi-scale model of the immune response following Mtb infection. We show that this model can capture various aspects of human and non-human primate TB disease and predict that biomarkers in the blood may only faithfully represent events in the lung at early time points after infection. We posit that HostSim, as a first step toward personalized digital twins in TB research, offers a powerful computational tool that can be used in concert with experimental approaches to understand and predict events about various aspects of TB disease and therapeutics.

category: systems biology


title: XomicsToModel: Multiomics data integration and generation of thermodynamically consistent metabolic models

abstract: Constraint-based modelling can mechanistically simulate the behaviour of a biochemical system, permitting hypotheses generation, experimental design and interpretation of experimental data, with numerous applications, including modelling of metabolism. Given a generic model, several methods have been developed to extract a context-specific, genome-scale metabolic model by incorporating information used to identify metabolic processes and gene activities in a given context. However, existing model extraction algorithms are unable to ensure that the context-specific model is thermodynamically feasible. This protocol introduces XomicsToModel, a semi-automated pipeline that integrates bibliomic, transcriptomic, proteomic, and metabolomic data with a generic genome-scale metabolic reconstruction, or model, to extract a context-specific, genome-scale metabolic model that is stoichiometrically, thermodynamically and flux consistent. The XomicsToModel pipeline is exemplified for extraction of a specific metabolic model from a generic metabolic model, but it enables multi-omic data integration and extraction of physicochemically consistent mechanistic models from any generic biochemical network. With all input data fully prepared, algorithmic completion of the pipeline takes ~10 min, however manual review of intermediate results may also be required, e.g., when inconsistent input data lead to an infeasible model.


title: A novel histone deacetylase inhibitor-based approach to eliminate microglia and retain astrocyte properties in glial cell culture.

abstract: The close association between astrocytes and microglia causes great difficulties to distinguish their individual roles in innate immune responses in central nervous system. Current chemical-based methods to eliminate microglia in glial cell culture introduce various molecular and functional alterations to astrocytes. Here, we describe a novel two-step approach to achieve a complete elimination of microglia without affecting the biological properties of co-cultured astrocytes by temporal treatment of histone deacetylase inhibitor trichostatin A (TSA). We verify TSA as a potent inducer for microglial-specific cell death, which also causes comprehensive gene expression changes in astrocytes. However, withdrawal of TSA not only ensures no microglia repopulation, but also restores all the gene expression changes in terms of astrocyte functions, including neurotrophic factors, glutamate and potassium transporters, and reactive astrocyte subtypes. By contrast, withdrawal of PLX5622, the commonly used colony-stimulating factor 1 receptor inhibitor neither prevents microglia repopulation nor restores the gene expression changes mentioned above. Using this method, we are able to discriminate differential roles of microglia and astrocytes in the induced expression of antiviral and pro-inflammatory cytokines upon various pathological stimuli including the spike protein of SARS-CoV-2. This simple and efficient method can be customized for the understanding of microglia-astrocyte interaction and the development of epigenetic therapies that target over-activated microglia in neuroinflammation-related diseases.


title: The P681H mutation in the Spike glycoprotein confers Type I interferon resistance in the SARS-CoV-2 alpha (B.1.1.7) variant

abstract: Variants of concern (VOCs) of severe acute respiratory syndrome coronavirus type-2 (SARS-CoV-2) threaten the global response to the COVID-19 pandemic. The alpha (B.1.1.7) variant appeared in the UK became dominant in Europe and North America in early 2021. The Spike glycoprotein of alpha has acquired a number mutations including the P681H mutation in the polybasic cleavage site that has been suggested to enhance Spike cleavage. Here, we show that the alpha Spike protein confers a level of resistance to the effects of interferon-{beta} (IFN{beta}) in lung epithelial cells. This correlates with resistance to restriction mediated by interferon-induced transmembrane protein-2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is necessary for comparative resistance to IFN{beta} in a molecularly cloned SARS-CoV-2 encoding alpha Spike. Overall, we suggest that in addition to adaptive immune escape, mutations associated with VOCs also confer replication advantage through adaptation to resist innate immunity.

category: microbiology


title: Long wavelength light that improves aged mitochondrial function selectively increases cytokine expression in serum and the retina

abstract:
Aged mitochondrial function can be improved with long wavelength light exposure. This reduces cellular markers of inflammation and can improve system function from fly though to human. Here, we ask what impact 670nm light has on cytokine expression using a 40 cytokine array in blood serum and retina in C57Bl6 mice. There was a relatively uniform increase in cytokine expression between 3 and 12 months of age in serum and retina.

670nm exposure was delivered daily for a week in 12 month old mice. This shifted patterns of cytokine expression in both serum and retina inducing a selective increase with some in serum increasing >5 fold. Changes in retina were smaller. In serum there were major increases in IL-7, 6, 13, 16 and 23, also in TNF- and CXCL 9 and 10. In retina the increases were found mainly in some IL (interleukins) and CXCLs (chemokines). A few cytokines were reduced by light exposure.

Changes in serum cytokines implies that long wavelengths impacts systemically even to unexposed tissues deep in the body. In the context of wider literature, increased cytokine expression may be protective. However, their upregulation by light merits further analysis as cytokines upregulation can also be negative.

category: molecular biology


title: LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

abstract:
Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host factors controlling SARS-CoV-2 Spike binding. The top hit was a Toll-like receptor-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where it forms a cell surface complex with LRRC15 but does not support infection. Instead, LRRC15 functioned as a negative receptor suppressing both pseudotyped and live SARS-CoV-2 infection. LRRC15 is expressed in collagen-producing lung myofibroblasts where it can sequester virus and reduce infection in trans. Mechanistically LRRC15 is regulated by TGF-{beta}, where moderate LRRC15 expression drives collagen production but high levels suppress it, revealing a novel lung fibrosis feedback circuit. Overall, LRRC15 is a master regulator of SARS-CoV-2, suppressing infection and controlling collagen production associated with "long-haul" COVID-19.

In BriefUsing pooled whole genome CRISPR activation screening, we identify the TLR relative LRRC15 as a novel SARS-CoV-2 Spike interacting protein. LRRC15 is not a SARS-CoV-2 entry receptor, but instead can suppress SARS-CoV-2 infection. LRRC15 is expressed by lung fibroblasts and regulates both collagen production and infection of ACE2-expressing target cells. This may provide a direct link between SARS-CoV-2 particles and lung fibrosis seen in "long-haul" COVID-19 patients.

HighlightsO_LIWhole genome CRISPR activation screening implicates the TLR relative LRRC15 in SARS-CoV-2 Spike binding
C_LIO_LILRRC15 suppresses live SARS-CoV-2 virus infection
C_LIO_LILRRC15 is expressed in lung fibroblasts and sequesters virus while controlling collagen production
C_LIO_LILRRC15 can act as a master regulator of infection and fibrosis, potentially controlling SARS-CoV-2 infection outcomes and "long-haul" COVID-19
C_LI


title: Potent antibody immunity to SARS-CoV-2 variants elicited by a third dose of inactivated vaccine

abstract: SARS-CoV-2 variants are still prevalent worldwide and continue to pose a challenge to the effectiveness of current vaccines. It remains unknown whether a third dose of inactivated vaccine elicits immune potential against SARS-CoV-2 variants. Here, we showed a significant decline in plasma neutralization against SARS-CoV-2 at seven months after a second dose of the inactivated vaccine in a large-scale cohort. However, we also found that a third vaccination with an inactivated vaccine largely increased plasma neutralization against variants including Beta, Delta, and Lambda. More importantly, the high-affinity anti-RBD memory B cells were also generated by the third vaccination, suggesting a more potent and longer protection. These findings highlighted the importance and effectiveness of a third dose of inactivated vaccine in conferring higher protection against the emerging variants in populations.


title: On-tissue spatial proteomics integrating MALDI-MS imaging with shotgun proteomics reveals soy consumption-induced biomarkers in a fragile X syndrome mouse model

abstract: Soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for unbiased protein biomarker identification in Fragile X syndrome (FXS) in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging (MSI) with label-free proteomics in a mouse model of FXS to map the spatial distribution and quantify the levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1,004 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in the GABAergic system, synaptic transmission, and co-expression network analysis indicated that protein in soy group was significantly associated with metabolism and synapse modules in the Fmr1KO brain. Ultimately, this spatial proteomics work laid the ground for identifying novel therapeutic targets and biomarkers for FXS.


title: Gender Imbalance in the Editorial Activities of a Researcher-led Journal

abstract: Editorial decision-making is a fundamental element of the scientific enterprise. We examined whether contributions to editorial decisions at various stages of the publication process is subject to gender disparity, based on analytics collected by the biomedical researcher-led journal eLife. Despite efforts to increase women representation, the board of reviewing editors (BRE) was men-dominant (69%). Moreover, authors suggested more men from the BRE pool, even after correcting for mens numerical over-representation. Although women editors were proportionally involved in the initial editorial process, they were under-engaged in editorial activities involving reviewers and authors. Additionally, converging evidence showed gender homophily in manuscripts assignment, such that men Senior Editors over-engaged men Reviewing Editors. This tendency was stronger in more gender-balanced scientific disciplines. Together, our findings confirm that gender disparities exist along the editorial process and suggest that merely increasing the proportion of women might not be sufficient to eliminate this bias.

category: scientific communication and education


title: tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

abstract: Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.


title: YAP1 Activation by Human Papillomavirus E7 Promotes Basal Cell Identity in Squamous Epithelia

abstract: Persistent human papillomavirus (HPV) infection of stratified squamous epithelial cells causes nearly five percent of cancer cases worldwide. HPV-positive oropharyngeal cancers harbor few mutations in the Hippo signaling pathway compared to HPV-negative cancers at the same anatomical site, prompting the hypothesis that an HPV-encoded protein inactivates the Hippo pathway and activates the Hippo effector YAP1. The HPV E7 oncoprotein is required for HPV infection and for HPV-mediated oncogenic transformation. We investigated the effects of HPV oncoproteins on YAP1 and found that E7 activates YAP1, promoting YAP1 nuclear localization in basal epithelial cells. YAP1 activation by HPV E7 required that E7 bind and degrade the tumor suppressor PTPN14. E7 required YAP1 transcriptional activity to extend the lifespan of primary keratinocytes, indicating that YAP1 activation contributes to E7 carcinogenic activity. Maintaining infection in basal cells is critical for HPV persistence, and here we demonstrate that YAP1 activation causes HPV E7 expressing cells to be retained in the basal compartment of stratified epithelia. We propose that YAP1 activation resulting from PTPN14 inactivation is an essential, targetable activity of the HPV E7 oncoprotein relevant to HPV infection and carcinogenesis.


title: Zymomonas mobilis ZM4 utilizes an acetaldehyde dehydrogenase to produce acetate

abstract:
Zymomonas mobilis is a promising bacterial host for biofuel production but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main byproducts generated by Z. mobilis is acetate but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetaldehyde dehydrogenase in Z. mobilis and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase.

ImportanceBiofuel production from non-food crops is an important strategy for reducing carbon emissions from the transportation industry but it has not yet become commercially viable. An important avenue to improve biofuel production is to enhance the characteristics of fermentation organisms by genetic engineering. To make genetic modifications successful, we must gain sufficient information about the genome and metabolism of the organism to enable rational design and engineering. Here, we improved understanding of Zymomonas mobilis, a promising biofuel producing bacterium, by identifying a metabolic pathway and associated gene that lead to byproduct formation. This information may be used in the future for genetic engineering to reduce byproduct formation during biofuel production.


title: Rapid antibiotic susceptibility testing and species identification for mixed infections

abstract: Antimicrobial resistance is an increasing problem globally. Rapid antibiotic susceptibility testing (AST) is urgently needed in the clinic to enable personalized prescription in high-resistance environments and limit the use of broad-spectrum drugs. Previously we have described a 30 min AST method based on imaging of individual bacterial cells. However, current phenotypic AST methods do not include species identification (ID), leaving time-consuming plating or culturing as the only available option when ID is needed to make the sensitivity call. Here we describe a method to perform phenotypic AST at the single-cell level in a microfluidic chip that allows subsequent genotyping by in situ FISH. By stratifying the phenotypic AST response on the species of individual cells, it is possible to determine the susceptibility profile for each species in a mixed infection sample in 1.5 h. In this proof-of-principle study, we demonstrate the operation with four antibiotics and a mixed sample with four species.


title: Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus

abstract: In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in Saudi Arabia and was mostly associated with severe respiratory illness in humans. Dromedary camels are the zoonotic reservoir for MERS-CoV. To investigate the biology of MERS-CoV in camelids, we developed a well-differentiated airway epithelial cell (AEC) culture model for Llama glama and Camelus bactrianus. Histological characterization revealed progressive epithelial cellular differentiation with well-resemblance to autologous ex vivo tissues. We demonstrate that MERS-CoV displays a divergent cell tropism and replication kinetics profile in both AEC models. Furthermore, we observed that in the camelid AEC models MERS-CoV replication can be inhibited by both type I and III interferons (IFNs). In conclusion, we successfully established camelid AEC cultures that recapitulate the in vivo airway epithelium and reflect MERS-CoV infection in vivo. In combination with human AEC cultures, this system allows detailed characterization of the molecular basis of MERS-CoV cross-species transmission in respiratory epithelium.


title: Selective clonal persistence of human retroviruses in vivo: radial chromatin organization, integration site and host transcription

abstract: The human retroviruses HTLV-1 and HIV-1 persist in vivo, despite the host immune response and antiretroviral therapy, as a reservoir of latently infected T-cell clones. It is poorly understood what determines which clones survive in the reservoir and which are lost. We compared >160,000 HTLV-1 integration sites from T-cells isolated ex vivo from naturally-infected subjects with >230,000 integration sites from in vitro infection, to identify the genomic features that determine selective clonal survival. Three factors explained >40% of the observed variance in clone survival of HTLV-1 in vivo: the radial intranuclear position of the provirus, its absolute genomic distance from the centromere, and the intensity of host genome transcription flanking the provirus. The radial intranuclear position of the provirus and its distance from the centromere also explained ~7% of clonal persistence of HIV-1 in vivo. Selection for transcriptionally repressive nuclear compartments favours clonal persistence of human retroviruses in vivo.


title: Immune escape mutations selected by neutralizing antibodies in natural HIV-1 infection can alter coreceptor usage repertoire of the transmitted/founder virus

abstract: The ability of HIV-1 to evade neutralizing antibodies (NAbs) in vivo is well demonstrated, but the impact of NAb escape mutations on HIV-1 phenotype other than immune escape itself has rarely been studied. Here, we show that immune escape mutations selected by V3-glycan specific NAbs in vivo can alter the coreceptor usage repertoire of the transmitted/founder (T/F) HIV-1. In a participant developed V3-glycan NAb response, naturally selected mutations at the V3 N301 and N332 glycan sites abrogated CCR8 usage while conferred APJ usage on the cognate T/F strain. Mutations at the N301 glycan also impaired CCR3 usage and partially compromised the efficiency in using CCR5, which could be fully restored by a single escape mutation at the N332 glycan site. Our study demonstrates the link between NAb escape and coreceptor usage alteration in natural HIV-1 infection and indicates that NAb response could drive virus entry tropism evolution in vivo.


title: Intrasexual cuticular hydrocarbon dimorphism in a wasp sheds light on hydrocarbon biosynthesis genes in Hymenoptera

abstract: Cuticular hydrocarbons (CHCs) cover the cuticle of insects and serve as desiccation barrier and for chemical communication. While the main enzymatic steps of CHC biosynthesis are well understood, few of the underlying genes have been identified. Here we show how exploitation of intrasexual CHC dimorphism in a mason wasp, Odynerus spinipes, in combination with whole-genome sequencing and comparative transcriptomics facilitated identification of such genes. RNAi-mediated knockdown of twelve candidate gene orthologs in honey bees, Apis mellifera, confirmed nine genes impacting CHC profile composition. Most of them have predicted functions consistent with current knowledge of CHC metabolism. However, we found first-time evidence for a fatty acid amide hydrolase also influencing CHC profile composition. In situ hybridization experiments furthermore suggest trophocytes participating in CHC biosynthesis. Our results set the base for experimental CHC profile manipulation in Hymenoptera and imply that the evolutionary origin of CHC biosynthesis predates the arthropods colonization of land.


title: Improvement of eukaryotic proteins prediction from soil metagenomes

abstract:
BackgroundDuring the last decades, shotgun metagenomics and metabarcoding have highlighted the diversity of microorganisms from environmental or host-associated samples. Most assembled metagenome public repositories use annotation pipelines tailored for prokaryotes regardless of the taxonomic origin of contigs and metagenome-assembled genomes (MAGs). Consequently, eukaryotic contigs and MAGs, with intrinsically different gene features, are not optimally annotated, resulting in an incorrect representation of the eukaryotic component of biodiversity, despite their biological relevance.

ResultsUsing an automated analysis pipeline, we have filtered 7.9 billion of contigs from 6,873 soil metagenomes in the IMG/M database of the Joint Genome Institute to identify eukaryotic contigs. We have re-annotated genes using eukaryote-tailored methods, yielding 8 million eukaryotic proteins. Of these, 5.6 million could be traced back to non-chimeric higher confidence eukaryotic contigs. Our pipeline improves eukaryotic proteins completeness, contiguity and quality. Moreover, the better quality of eukaryotic proteins combined with a more comprehensive assignment method improves the taxonomic annotation as well.

ConclusionsUsing public soil metagenomic data, we provide a dataset of eukaryotic soil proteins with improved completeness and quality as well as a more reliable taxonomic annotation. This unique resource is of interest for any scientist aiming at studying the composition, biological functions and gene flux in soil communities involving eukaryotes.

O_TEXTBOXKey Points

O_LIEukaryotic micro-organisms have key roles in soil microbial communities and ecosystems.
C_LIO_LIEukaryotic genes and proteins are incorrectly represented in metagenomic libraries due to inappropriate methods dedicated to prokaryotes.
C_LIO_LIWe have improved the quality and completeness of the de novo proteins prediction and taxonomic annotation of eukaryotic organisms from soil or plant-associated microbiome.
C_LI

C_TEXTBOX


title: In silico Analysis of Nonsynonymous Genomic Variants within CCM2 Gene Reaffirm the Existence of Dual Cores within Typical PTB Domain

abstract:
PurposeThe objective of this study is to validate the existence of dual cores within the typical phosphotyrosine binding (PTB) domain and to identify potentially damaging and pathogenic nonsynonymous coding single nuclear polymorphisms (nsSNPs) in the canonical PTB domain of the CCM2 gene that causes cerebral cavernous malformations (CCMs).

MethodsThe nsSNPs within the coding sequence for PTB domain of human CCM2 gene, retrieved from exclusive database search, were analyzed for their functional and structural impact using a series of bioinformatic tools. The effects of the mutations on tertiary structure of the PTB domain in human CCM2 protein were predicted to examine the effect of the nsSNPs on tertiary structure on PTB Cores.

ResultsOur mutation analysis, through alignment of protein structures between wildtype CCM2 and mutant, indicated that the structural impacts of pathogenic nsSNPs is biophysically limited to only the spatially adjacent substituted amino acid site with minimal structural influence on the adjacent core of the PTB domain, suggesting both cores are independently functional and essential for proper CCM2 function.

ConclusionUtilizing a combination of protein conservation and structure-based analysis, we analyzed the structural effects of inherited pathogenic mutations within the CCM2 PTB domain. Our results indicated that the pathogenic amino acid substitutions lead to only subtle changes locally confined to the surrounding tertiary structure of the PTB core within which it resides, while no structural disturbance to the neighboring PTB core was observed, reaffirming the presence of dual functional cores in the PTB domain.


title: An evolutionary divergent thermodynamic brake in ZAP-70 finetunes the kinetic proofreading in T cell

abstract: T cell signaling starts with assembling several tyrosine kinases and adaptor proteins to the T cell receptor (TCR), following the antigen binding. The lifetime of the TCR: antigen complex and the time delay between the recruitment and activation of each kinase determines the T cell response. The mechanism by which the time delays are implemented in TCR signaling is not fully understood. Combining experiments and kinetic modeling, we here report a thermodynamic-brake in the regulatory module of ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation in TCR. Phylogenetic analysis revealed that the evolution of the thermodynamic-brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. Paralogous kinase Syk expressed in B cells, does not possess such a functional thermodynamic brake, which may explain higher basal activation and lack of ligand selectivity by Syk.


title: Soft, dynamic hydrogel confinement improves kidney organoid lumen morphology and reduces epithelial-mesenchymal transition in culture

abstract: Pluripotent stem cell-derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off-target cells and phenotypic alterations, preventing maturity. Here, we created various dynamic hydrogel architectures, conferring a controlled and biomimetic environment for organoid encapsulation. We investigated how hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers. We observed stiff hydrogel encapsulation led to an absence of certain renal cell types and signs of an epithelial- mesenchymal transition (EMT), whereas encapsulation in soft-stress-relaxing hydrogels led to all major renal segments, fewer fibrosis/EMT associated proteins, apical proximal tubule enrichment, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. Our findings show that engineering hydrogel mechanics and dynamics has a decided benefit for organoid culture. These structure-property- function relationships can enable rational design of materials, bringing us closer to functional engraftments and disease-modelling applications.

category: bioengineering


title: Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

abstract:
BackgroundA common polymorphism (R577X) in the ACTN3 gene results in complete absence of the Z-disc protein -actinin-3 from fast-twitch muscle fibres in ~16% of the worlds population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 30% stretch produced a significant ~40% deficit in maximum force; here we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this.

MethodsSingle fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means {+/-} SD and tested for significance using ANOVA.

ResultsWe show that the absence of -actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts.

ConclusionsAbsence of -actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that, in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0+ 30%, but not, L0+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why -actinin-3 deficient humans have been reported to have a faster decline in muscle function with increasing age, that is; as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional -actinin-3 deficient fibres will be increased, resulting in fibres breakages.


title: Newly recruited intraepithelial Ly6A+CCR9+CD4+ T cells protect against enteric viral infection

abstract: The intestinal epithelium comprises the bodys largest surface exposed to viruses. However, a role for intraepithelial T lymphocytes in resistance against viral infections remain elusive. By fate-mapping T cells recruited to the murine intestinal epithelium, we observed accumulation of CD4+ T cells after infection with murine norovirus (MNV) or mouse adenovirus type-2 (AdV), but not after reovirus infection. Intraepithelial CD4+ T cells recruited after MNV or AdV infection co-express Ly6A and CCR9, and exhibit T helper 1 and cytotoxic profiles. Although these cells display a diverse TCR repertoire, they conferred protection against AdV and MNV both in vivo and in an organoid co-culture model in an IFN-{gamma}-dependent manner. Ablation of the T cell receptor (TCR) or the transcription factor ThPOK in CD4+ T cells prior to infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ T cells against enteric viruses.


title: Human gut derived Anaerotruncus colihominis ameliorates experimental autoimmune encephalomyelitis

abstract: The gut microbiome plays an important role in autoimmunity including multiple sclerosis and its mouse model called experimental autoimmune encephalomyelitis (EAE). The gut-brain axis refers to the complex interactions between the gut microbiota and the nervous and immune systems linking brain and gut functions. Prior studies have demonstrated that the multiple sclerosis gut microbiota can contribute to disease hence making it a potential therapeutic target. Other studies have reported that long-term antibiotic therapy in multiple sclerosis patients reduces relapse rate and gadolinium enhancing lesions as well as improves measures of disability. In addition, antibiotic treatment has been shown to ameliorate disease in the EAE mouse model of multiple sclerosis. Yet, to this date, the mechanisms mediating these antibiotics effects are not understood. Furthermore, there is no consensus on the gut derived bacterial strains that drive neuroinflammation in multiple sclerosis. Hence, it remains unclear how the gut microbiota can be targeted for therapeutic purposes in multiple sclerosis patients. Here we characterized the gut microbiome of untreated and vancomycin treated EAE mice over time to identify bacteria with neuroimmunomodulatory potential. We observed alterations in the gut microbiota composition following EAE induction. We found that vancomycin treatment ameliorates EAE and that this protective effect is mediated via the microbiota. Notably, we observed increase abundance of bacteria known to be strong inducers of regulatory T cells including members of Clostridium clusters XIVa and XVIII in vancomycin-treated mice during the presymptomatic phase of EAE as well as at disease peak. We identified 50 bacterial taxa that correlate with EAE severity. Interestingly, several of these taxa exist in the human gut and some of them have been implicated in multiple sclerosis including Anaerotruncus colihominis which had a positive correlation with disease severity. Unexpectedly, we found that Anaerotruncus colihominis ameliorates EAE and this is associated with induction of ROR{gamma}t+ regulatory T cells in the mesenteric lymph nodes. Together, our results identify vancomycin as a potent modulator of the gut-brain axis by promoting the proliferation of bacterial species that induce regulatory T cells. In addition, our findings reveal 50 gut commensals as regulator of the gut-brain axis that can be used to further characterize pathogenic and beneficial hostmicrobiota interactions in multiple sclerosis patients. Our findings suggest that elevated Anaerotruncus colihominis in multiple sclerosis patients may represent a protective mechanism associated with recovery from the disease.


title: Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing

abstract: The mRNA 5 cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-encoding genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.


title: Predicting efficiency of writing short sequences into the genome using prime editing

abstract: Short sequences can be precisely written into a selected genomic target using prime editing. This ability facilitates protein tagging, correction of pathogenic deletions, and many other exciting applications. However, it remains unclear what types of sequences prime editors can easily insert, and how to choose optimal reagents for a desired outcome. To characterize features that influence insertion efficiency, we designed a library of 2,666 sequences up to 69 nt in length and measured the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems. We discover that insertion sequence length, nucleotide composition and secondary structure all affect insertion rates, and that mismatch repair proficiency is a strong determinant for the shortest insertions. Combining the sequence and repair features into a machine learning model, we can predict insertion frequency for new sequences with R = 0.69. The tools we provide allow users to choose optimal constructs for DNA insertion using prime editing.


title: Deep assessment of human disease-associated ribosomal RNA modifications using Nanopore direct RNA sequencing

abstract: The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridines, 2-O-methylation of riboses and acetylation of cytidines. Targeted direct rRNA-seq analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.


title: Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease

abstract:
MYH9-related disease patients with mutations in the contractile protein non-muscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild to moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in MYH9-related disease patients, and treatment with tranexamic acid can improve the hemostatic function.

TeaserImpaired hemostasis in Myh9 mutant mice due to reduced platelet forces can be improved by tranexamic acid.


title: Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain specific lifespan and health effects in Caenorhabditis nematodes

abstract: The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies--for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions.


title: Quantitative analysis of transcription start site selection in Saccharomyces cerevisiae determines contributions of DNA sequence and RNA Polymerase II activity

abstract: DNA sequence at Transcription Start Sites (TSSs) is a key determinant of initiation by RNA Polymerase II (Pol II). To function as a TSS, an initiation compatible sequence must be specified by a promoter in an appropriate chromatin context. We report the development of a method for quantitative analysis of transcription initiation by Pol II that involves construction of DNA libraries of barcoded promoter variants, production of RNA transcripts, and analysis of transcript 5 ends and transcript yields (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of transcription initiation during "promoter scanning" by Saccharomyces cerevisiae Pol II for ~1 million unique TSS sequences. Furthermore, we employ Pol II MASTER to determine how Pol II activity, known to widely alter TSS selection in vivo, alters TSS efficiencies across our promoter variants. Pol II MASTER recapitulates known critical qualities of Saccharomyces cerevisiae TSS -8, -1, and +1 positions while demonstrating that surrounding sequences modulate initiation efficiency over a wide range. We discover functional interactions between neighboring sequence positions, indicating that adjacent positions likely function together. We demonstrate that initiation efficiencies are altered for +1 A TSSs relative to +1 G TSSs when Pol II activity is perturbed through genetic means. Pol II MASTER provides data for predictive models of TSS initiation efficiency at genomic promoters.


title: A theory of the dynamics of DNA loop initiation in condensin/cohesin complexes

abstract: The structural maintenance of chromosome complexes exhibit the remarkable ability to actively extrude DNA, which has led to the appealing and popular "loop extrusion" model to explain one of the most important processes in biology: the compaction of chromatin during the cell cycle. A potential mechanism for the action of extrusion is the classic Brownian ratchet, which requires short DNA loops to overcome an initial enthalpic barrier to bending, before favoured entropic growth of longer loops. We present a simple model of the constrained dynamics of DNA loop formation based on a frictional worm like chain, where for circular loops of order, or smaller than the persistence length, internal friction to bending dominates solvent dynamics. Using Rayleighs dissipation function, we show how bending friction can be translated to simple one dimensional diffusion of the angle of the loop resulting in a Smoluchowski equation with a coordinate dependent diffusion constant. This interplay between Brownian motion, bending dissipation and geometry of loops leads to a qualitatively new phenomenon, where the friction vanishes for bends with an angle of exactly 180 degrees, due to a decoupling between changes in loop curvature and angle. Using this theory and given current parameter uncertainties, we tentatively predict mean first passage times of between 1 and 10 seconds, which is of order the cycle time of ATP, suggesting spontaneous looping could be sufficient to achieve efficient initiation of looping.

category: biophysics


title: Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1

abstract:
The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming 1S subunit and the auxiliary 2{delta}-1 and {gamma}1 subunits. Particularly, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage-dependence of activation to positive potentials outside the physiological range. We generated a new HEK293-cell line stably expressing 2{delta}-1, {beta}3, and STAC3. When the adult (CaV1.1a) and the embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage-dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further co-expressed the {gamma}1 subunit, the current density of CaV1.1a, but not of CaV1.1e, was reduced by >50 %. In addition, {gamma}1 caused a shift of the voltage-dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of {gamma}1, but not its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between oppositely charged residues in the IVS3-S4 loop and the {gamma}1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the {gamma}1-dependent reduction of current density, suggesting that structural rearrangements of CaV1.1a induced by inclusion of exon 29 allosterically empower the {gamma}1 subunit to exert its inhibitory action on CaV1.1 calcium currents.

SummaryEl Ghaleb et al. analyzed the effects of the {gamma}1 subunit on current properties and expression of the adult (CaV1.1a) and embryonic (CaV1.1e) calcium channel splice variants, demonstrating that {gamma}1 reduces the current amplitude in a splicing-dependent manner.


title: Aspergillus fumigatus drives tissue damage via iterative assaults upon mucosal integrity and immune homeostasis

abstract:
The human lung is constantly exposed to Aspergillus fumigatus spores, the most prevalent worldwide cause of fungal respiratory disease. Pulmonary tissue damage is a unifying feature of Aspergillus-related diseases; however, the mechanistic basis of damage is not understood. In the lungs of susceptible hosts A. fumigatus undergoes an obligatory morphological switch involving spore germination and hyphal growth. We modelled A. fumigatus infection in cultured A549 human pneumocytes, capturing phosphoactivation status of five host signalling pathways, nuclear translocation & DNA binding of eight host transcription factors, and expression of nine host response proteins over six time points encompassing exposures to live fungus and the secretome thereof. The resulting dataset, comprised of more than 1000 data points, reveals that pneumocytes mount differential responses to A. fumigatus spores, hyphae and soluble secreted products via the NF-kB, JNK, and JNK + p38 pathways respectively. Importantly, via selective degradation of host pro-inflammatory (IL-6 and IL-8) cytokines and growth factors (FGF-2), fungal secreted products reorchestrate the host response to fungal challenge as well as driving multiparametric epithelial damage, culminating in cytolysis. Dysregulation of NF-kB signalling, involving iterative stimulation of canonical and non-canonical signalling, was identified as a significant feature of host damage both in vitro and in a mouse model of invasive aspergillosis. Our data demonstrate that composite tissue damage results from iterative exposures to different fungal morphotypes and secreted products and suggest that modulation of host responses to fungal challenge might represent a unified strategy for therapeutic control of pathologically distinct types of Aspergillus-related disease.

IMPORTANCEPulmonary aspergillosis is a spectrum of diseases caused primarily by Aspergillus fumigatus. This fungus is ubiquitous in the environment and grows as a mold, which harbors and disperses spores into the environment. Like other airborne pathogens, the lung mucosa is the first point of contact with the fungus post inhalation. The outcome and severity of disease depends on the host-fungal interaction at the lung interface. We studied how the human lung interacts with spore, germ tube and hyphae growth forms to understand the sequence and dynamics of the early events, which are critical drivers of disease development and progression. Our work is significant in identifying, in response to fungal secreted products, non-canonical NF-kB activation via RelB as being a driving factor in fungus-mediated lung damage. This process could be modulated therapeutically to protect the integrity of infected lung mucosae.


title: Photoimmunotherapy retains its anti-tumor efficacy with increasing stromal content in heterotypic pancreatic cancer spheroids

abstract: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by increased levels of desmoplasia that contributes to reduced drug delivery and poor treatment outcomes. In PDAC, the stromal content can account for up to 90% of the total tumor volume. The complex interplay between stromal components, including pancreatic cancer associated fibroblasts (PCAFs), and PDAC cells in the tumor microenvironment (TME) have a significant impact on prognoses and thus needs to be recapitulated in vitro when evaluating various treatment strategies. This study is a systematic evaluation of photodynamic therapy (PDT) in 3D heterotypic coculture models of PDAC with varying ratios of patient derived PCAFs that simulate heterogenous PDAC tumors with increasing stromal content. The efficacy of antibody-targeted PDT (photoimmunotherapy; PIT) using cetuximab photoimmunoconjugates (PICs) of benzoporphyrin derivative (BPD) is contrasted with that of liposomal BPD (Visudyne(R)), which is currently in PDT clinical trials for PDAC. We demonstrate that both Visudyne(R)-PDT and PIT were effective in heterotypic PDAC 3D spheroids with a low stromal content. However, as the stromal content increases above 50% in the 3D spheroids, the efficacy of Visudyne(R)-PDT is reduced by up to 10-fold, while PIT retains its efficacy. PIT was found to be 10-fold, 19-fold and 14-fold more phototoxic in spheroids with 50%, 75% and 90% PCAFs, respectively, as compared to Visudyne(R)-PDT. This marked difference in efficacy is attributed to the ability of PICs to penetrate and distribute within spheroids with a higher stromal content, whereas Visudyne(R) is restricted to the spheroid periphery. This study thus demonstrates how the stromal content in PDAC spheroids directly impacts their responsiveness to PDT and proposes PIT to be a highly suited treatment option for desmoplastic tumors with particularly high degrees of stromal content.


title: Vitamin D deficiency increases prostatic megalin expression and globulin-bound testosterone import, increasing prostatic androgens in African American men

abstract: Vitamin D deficiency associates with an increased risk of prostate cancer (PCa) mortality and is hypothesized to contribute to PCa aggressiveness and disparities in African Americans. We previously reported a relationship between African-ancestry, circulating and intraprostatic vitamin D metabolites and prostatic expression of megalin, an endocytic receptor that internalizes globulin-bound hormones. Here, we show prostatic 5-dihydrotestosterone (DHT) levels are higher in African American men and inversely correlate with serum 25-hydroxyvitamin D (25D) status. We further demonstrate that megalin imports sex hormone-binding globulin-bound testosterone in prostate cells and prostatic loss of Lrp2 (megalin) results in reduced prostate androgen levels. Megalin expression was suppressed by 25D and DHT in cell lines, patient-derived prostate epithelial cells, and prostate tissue explants, supporting a negative feedback loop. Megalin levels are reduced in localized PCa by Gleason Grade and in patients with future disease recurrence. Our findings highlight the impact of vitamin D deficiency on prostate androgen levels, which are known drivers of PCa, and reveal a potential mechanistic link between vitamin D and the PCa disparities observed in African Americans.


title: Prostate megalin is regulated by serum hormone levels and imports globulin-bound testosterone


title: Recurrent activating mutations in AHR act as drivers of urinary tract cancer

abstract: Bladder cancer has a high recurrence rate and low survival of advanced stage patients. Few genetic drivers of bladder cancer have thus far been identified. We performed in-depth structural variant analysis on whole-genome sequencing data of 206 metastasized urinary tract cancers. In ~10% of the patients, we identified recurrent in-frame deletions of exons 8 and 9 in the aryl hydrocarbon receptor gene (AHR{Delta}e8-9), which codes for a ligand-activated transcription factor. Pan-cancer analyses show that AHR{Delta}e8-9 is highly specific to urinary tract cancer and mutually exclusive with other bladder cancer drivers. In AHR{Delta}e8-9 the ligand-binding domain is disrupted and we show that this results in ligand-independent AHR-pathway activation. In bladder organoids, AHR{Delta}e8-9 induces a transformed phenotype that is characterized by upregulation of AHR target genes, downregulation of differentiation markers and upregulation of genes associated with stemness and urothelial cancer. Furthermore, AHR{Delta}e8-9 expression results in anchorage independent growth of bladder organoids, indicating tumorigenic potential. DNA-binding deficient AHR{Delta}e8-9 fails to induce transformation, suggesting a role for AHR target genes in the acquisition of the oncogenic phenotype. In conclusion, we show that AHR{Delta}e8-9 is a novel driver of urinary tract cancer and that the AHR pathway could be an interesting therapeutic target.


title: Recurrent exon-deleting activating mutations in AHR act as drivers of urinary tract cancer