title: PCA outperforms popular hidden variable inference methods for QTL mapping

abstract: Estimating and accounting for hidden variables is widely practiced as an important step in quantitative trait locus (QTL) analysis for improving the power of QTL identification. Here we benchmark popular hidden variable inference methods including surrogate variable analysis (SVA), probabilistic estimation of expression residuals (PEER), and hidden covariates with prior (HCP) against principal component analysis (PCA; a well-established dimension reduction and factor discovery method) through comprehensive simulation studies and show that PCA is faster and better-performing. In particular, we show that under realistic simulation scenarios, PEER--the most popular hidden variable inference method for QTL mapping to date--does not have the alleged advantage that its performance does not deteriorate as the number of inferred covariates increases. Additionally, our real data analysis shows that PEER produces identical results as PCA at best, is at least three orders of magnitude slower, and can fail to capture important variance components of the molecular phenotype data in certain cases. Based on these results and the fact that PCA is much easier to interpret and use, we contend that PCA should be the preferred hidden variable inference method for QTL mapping until evidence suggests otherwise. To help researchers use PCA in their QTL analysis, we provide an R package PCAForQTL along with a detailed tutorial.

category: bioinformatics


title: Clinical application of tumour in normal contamination assessment from whole genome sequencing

abstract: Bioinformatics pipelines designed for paired analysis of tumour and matched normal samples use germline variants detected from normal tissue samples to identify bona fide tumour somatic mutations. Unexpected contamination of normal samples with tumour cells can lead to the subtraction of genuine somatic variants, reducing variant detection sensitivity and compromising downstream analyses. Leveraging the power of whole-genome sequencing data available from the 100,000 Genomes Project at Genomics England, we developed the Tumour in Normal Contamination (TINC) tool for TIN contamination assessment. TINC was validated in silico using patient-derived whole-genome samples (R2 = 0.95; p < 2.2 x 10-16 and R2 = 0.85; p < 3. 3 x 10-13 for expected versus observed contamination in two tumour types), and against orthogonal minimal residual disease (MRD) testing with 70 leukaemia samples. From a systematic review of whole-genome data available for 771 patients with haematological malignancies and sarcomas, we found TIN contamination (>1%) across a range of cancer clinical indications and DNA sources. The highest prevalence was among saliva samples from acute myeloid leukaemia (AML) patients (43/114 samples, 38%), and sorted CD3+ T cells from myeloproliferative neoplasms (MPN) patients (22/24 samples, 91%). Further exploration of genomic data for AML and MPN patients has revealed 108 hotspot mutations in genes with high prevalence of somatic alterations in haematological cancers (JAK2, FLT3, DNMT3A, TP53, KIT, NRAS and IDH2), 27 of which supported by at least 5% of reads in the normal sample and therefore at risk of being subtracted by standard pipeline for somatic variant calling. Our work highlights the importance of contamination assessment for accurate detection of somatic variants in both research and clinical settings. TINC allows accurate and highly sensitive quantification of contamination levels, giving confidence in the validity of the whole genome assessment of the tumour. We propose this to become an essential metric in clinic practice alongside other routine quality control measures, especially as large-scale pan-cancer sequencing projects are adopted to deliver accurate data from which to make clinical decisions for patient care.


title: Dynamic Network Curvature Analysis of RNA-Seq Data in Sarcoma

abstract: In this work, we utilized network features of cancer gene interactomes to cluster pediatric sarcoma tumors and identify candidate therapeutic targets in an unsupervised manner. RNA-Seq data were mapped to protein-level interactomes to construct weighted networks for mathematical analysis. We employed a geometric approach centered on a discrete notion of curvature, which provides a measure of the functional association between genes in the context of their connectivity. Specifically, we adopted a recently proposed dynamic extension of graph curvature to extract features of the non-Euclidean, multiscale structure of genomic networks. We propose a hierarchical clustering approach to reveal preferential gene clustering according to their geometric cooperation which captured the characteristic EWSR1-FLI1 fusion in Ewing sarcoma. We also performed in silico edge perturbations to assess systemic response to simulated interventions quantified by changes in curvature. These results demonstrate that geometric network-based features can be useful for identifying non-trivial gene associations in an agnostic manner.


title: Tension Gauge Tethers as tension-dependent lifetime sensors

abstract:
Mechanotransduction of cells, a time-taking process, relies on responding to tension transmitted along various supramolecular linkages. The minimal lifetimes of the linkages needed for proper mechanotransduction remain poorly understood. We propose that such information can be provided using tension-dependent lifetime sensors that undergo irreversible tension-dependent dissociation. We quantified the tension-dependent lifetime of DNA-based Tension Gauge Tethers. Comparing it with the observations from previous cell studies, the minimal lifetime of individual integrin-RGD linkage needed for cell spreading was determined to be tens of seconds over the physiological tension range.

PACS numbers: 87.14.gk, 87.15.-v, 87.15.He, 87.15.La, 87.80.-y

category: biophysics


title: Microscope Alignment Using Real-Time Imaging FCS

abstract:
AO_SCPLOWBSTRACTC_SCPLOWModern EMCCD and sCMOS cameras read out fluorescence data with single-molecule sensitivity at a rate of thousands of frames per second. Exploiting these capabilities in full requires data evaluation in real-time. The direct camera-read-out tool presented here allows access to the data while the camera is recording. This provides simplified and accurate alignment procedures for Total Internal Reflection and Light Sheet Fluorescence Microscopy (TIRFM, LSFM), and simplifies and accelerates fluorescence experiments. The tool handles a range of widely used EMCCD and sCMOS cameras and uses Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) for its evaluation. It is easily extendable to other camera models and other techniques and is a base for automated TIRMF and LSFM data acquisition.

SignificanceWe developed a direct camera readout (DCR) software tool that allows access to camera data during acquisition and provides real-time Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) analysis. DCR displays live feedback and due to the sensitivity of correlation analysis provides a sensitive tool for microscope alignment using simple solutions of fluorescent particles. DCR is adaptable to different cameras and evaluation strategies, reduces alignment time, accelerates experiments, and can be used for automation.


title: Thermodynamic stabilization of the von Willebrand Factor A1 domain due to loss-of-function disease-related mutations.

abstract: The von Willebrand disease (vWD) is the most common hereditary bleeding disorder, caused by defects of the von Willebrand Factor (vWF), a large extracellular protein in charge of adhering platelets at sites of vascular lesion. vWF carries out this essential homeostatic task, via the specific protein-protein interaction between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIB). Upon the vWF activation triggered by the shear of the flowing blood. The two naturally occurring mutations G1324A and G1324S at the A1 domain, near the GPIB binding site, result in a dramatic decrease of platelets adhesion, a bleeding disorder classified as type 2M vWD. However, it remained unclear how these two supposedly minor modifications lead to this drastic phenotypic response. We addressed this question using a combination of equilibrium-molecular dynamics (MD) and non-equilibrium MD-based free energy simulations. Our data confirm that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. These mutations locally diminished the flexibility of the binding site to GPIB and induced a conformational change that affected the nearby secondary structure elements. Furthermore, we observed two significant changes in the vWF A1 domain upon mutation, the global redistribution of the internal mechanical stress and the increased thermodynamic stability of the A1 domain. These observations are consistent with previously-reported mutation-augmented melting temperatures. Overall, our results support the idea of thermodynamic conformational restriction of A1-- before the binding to GPIB--as a crucial factor determining the loss-of-function of the G1324A(S) vWD mutants.


title: Allosteric inhibitors of Zika virus NS2B-NS3 protease targeting protease in super-open conformation

abstract: Zika virus (ZIKV) is a member of the Flaviviridae family and is considered a major health threat causing cases of microcephaly in newborns and Guillain-Barre syndrome in adults. Here we targeted a transient deep and hydrophobic pocket of the super-open conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the orthosteric inhibitors. After virtual docking screening of approximately 7 million compounds against the novel allosteric site we selected the top seven candidates and tested them in an enzymatic assay. Six out of seven top candidates selected by the docking screen inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations, as well as suppressing viral replication. These six compounds, targeting the selected protease pocket conserved in ZIKV as well as several other Flaviviruses, have opened an opportunity for a new kind of drug candidate that might be useful to treat several flaviviral infections.

category: pharmacology and toxicology


title: Identification and quantification of α- and β-amanitin in wild mushrooms by HPLC-UV-EC and HPLC-DAD-MS detection

abstract:
Amatoxins are a group of highly toxic peptides, which include - and {beta}-amanitin, found in several species of mushrooms (e.g. Amanita phalloides). Due to their high hepatotoxicity, they account for most deaths occurring after mushrooms ingestion. The determination of - and {beta}- amanitin content in wild mushrooms is invaluable for treating cases involving poisoning. In the present study, we have developed and validated an analytical method based on high-performance liquid chromatography, with in-line ultraviolet and electrochemical detection (HPLC-UV-EC), for the rapid quantification of - and {beta}-amanitin in wild mushroom samples collected from the Inner Center of Portugal. A reproducible and simple solid-phase extraction (SPE) using OASIS(R) PRIME HLB cartridges was used for sample pre-treatment, followed by chromatographic separation based on the RP-C18 column. The UV and EC chromatograms of - and {beta}-amanitin were recorded at 305 nm and +0.600 V vs. Ag/AgCl, respectively. The linear quantification for both amanitins was in the range of 0.5-20.0 g{middle dot}mL-1 (R2 > 0.999). The LOD, calculated based on the calibration curve, was similar for UV and EC detection (0.12-0.33 g ml.-1). Intra-day and inter-day precision were less than 13%, and the recovery ratios ranged from 89% to 117%. Nine Amanita species and five edible mushrooms were analysed by HPLC-UV-EC, and HPLC-DAD-MS confirmed the identification of amatoxins. We find high - and {beta}-amanitin content in A. phalloides and not in the other species analysed. In sum, the developed and validated method provides a simple and fast analysis of - and {beta}-amanitins contents in wild mushrooms and is suitable for screening and routine assessment of mushroom intoxication.

HighlightsO_LINew validated method using HPLC-UV-EC to determine - and {beta}-amanitin in wild mushrooms.
C_LIO_LIReproducible and fast SPE procedure for small samples.
C_LIO_LIEffective sample pre-treatment with the OASIS(R) PRIME HLB SPE cartridge.
C_LIO_LIIdentification and quantification of - and {beta}-amanitin in wild mushroom samples from Portugal.
C_LIO_LIHPLC-DAD-MS confirmation of amatoxins present in mushroom samples.
C_LI

Graphical abstract

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title: A WAX INDUCER1/SHINE transcription factor controls cuticular wax in barley

abstract: All land plants seal their above ground body parts with a lipid-rich hydrophobic barrier called the cuticle that protects tissues from dehydration and other terrestrial threats. Mutational studies in several model species, including barley, have resolved multiple loci regulating cuticular metabolism and development. Of particular importance are the eceriferum (cer) mutants characterized by visual alterations in cuticular wax. In barley, some cer mutants, such as cer-x lines, show defects in the distinctive {beta}-diketone-enriched wax bloom on reproductive stage leaf sheaths, stems and spikes. In our study we exploited extensive allelic populations, near-isogenic lines and powerful genotyping platforms to identify variation in the HvWAX INDUCER1 (HvWIN1) gene as causal for cer-x mutants. We show that HvWIN1 function reduces cuticular permeability, promotes the accumulation of {beta}-diketones, and regulates cuticular metabolic gene expression. Analyses across the barley pangenome and hundreds of exome-capture datasets revealed high sequence conservation of HvWIN1 but also two non-synonymous variants exclusive to cultivated germplasm. Taken together, we suggest that variation in HvWIN1 controls multiple cuticular features in barley by controlling the expression of genes involved in cuticle development.

category: plant biology


title: Programming bacteria to sense environmental DNA for multiplexed pathogen detection

abstract: DNA is a universal and programmable signal of living organisms. Here we developed Cell-based DNA sensors (Cell-Sens) by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity and sensitivity. Multiplexed detection of combinations of different species genomic DNA was achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We demonstrate that the DNA sensors can detect presence of species in a microbial community without requiring DNA purification of the donor species. In sum, the modularity of the living cell-based DNA sensing mechanism and simple measurement procedure could enable programmable DNA sensing for broad applications.

category: synthetic biology


title: Quantitative Systems Pharmacology Modeling Framework of Autophagy in Tuberculosis: Application to Adjunctive Metformin Host-Directed Therapy

abstract:
BackgroundQuantitative systems pharmacology (QSP) modeling of the host-immune response against Mtb can inform rational design of host-directed therapies (HDTs). We aimed to develop a QSP framework to evaluate the effects of metformin-associated autophagy-induction in combination with antibiotics.

MethodsA QSP framework for autophagy was developed by extending a model for host-immune response to include AMPK-mTOR-autophagy signalling. This model was combined with pharmacokinetic-pharmacodynamic models for metformin and antibiotics against Mtb. We compared the model predictions to mice infection experiments, and derived predictions for pathogen and host-associated dynamics in humans treated with metformin in combination with antibiotics.

ResultsThe model adequately captured the observed bacterial load dynamics in mice Mtb infection models treated with metformin. Simulations for adjunctive metformin therapy in newly diagnosed patients suggested a limited yet dose-dependent effect of metformin on reducing the intracellular bacterial load and selected pro-inflammatory cytokines. Our predictions suggest that metformin may provide beneficiary effects when overall bacterial load, or extracellular-to-intracellular bacterial ratio is low, either early after infection or late during antibiotic treatment.

ConclusionsWe present the first QSP framework for HDTs against Mtb, linking cellular-level autophagy effects to disease progression. This framework may be extended to guide design of HDTs against Mtb.

category: systems biology


title: Comparative transcriptomics of the Drosophila olfactory subsystems identifies a support cell-expressed Osiris protein required for pheromone sensing

abstract: The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae comprise two morphologically and functionally distinct subsystems that express Odorant receptors (Ors) or Ionotropic receptors (Irs). Although these receptors have been thoroughly characterized in this species, the subsystem-specific expression and roles of other genes are much less well-understood. Here we generate subsystem-specific transcriptomic datasets to identify hundreds of genes, encoding diverse protein classes, that are selectively enriched in either Or or Ir subsystems. Using single-cell antennal transcriptomic data and RNA in situ hybridization, we find most neuronal genes - other than sensory receptor genes - are broadly expressed within the subsystems. By contrast, we identify many non-neuronal genes that exhibit highly selective cell-type expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of these olfactory subsystems. We characterize one Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large family of insect transmembrane proteins. Osi8 is expressed in tormogen support cells that are associated with pheromone sensing neurons. Loss of Osi8 abolishes high sensitivity neuronal responses to pheromone ligands. Together this work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in sensory responses, and provides a resource for future characterization of other olfactory subsystem-specific genes.

category: neuroscience


title: Cognitive penetrability of scene representations based on horizontal image disparities

abstract: The structure of natural scenes is signaled by many visual cues. Principal amongst them are the binocular disparities created by the laterally separated viewpoints of the two eyes. Disparity cues are believed to be processed hierarchically, first in terms of local measurements of absolute disparity and second in terms of more global measurements of relative disparity that allow extraction of the depth structure of a scene. Psychophysical and oculomotor studies have suggested that relative disparities are particularly relevant to perception, whilst absolute disparities are not. Here, we compare neural responses to stimuli that isolate the absolute disparity cue with stimuli that contain additional relative disparity cues, using the high temporal resolution of EEG to determine the temporal order of absolute and relative disparity processing. By varying the observers task, we assess the extent to which each cue is cognitively penetrable. We find that absolute disparity is extracted before relative disparity, and that task effects arise only at or after the extraction of relative disparity. Our results indicate a hierarchy of disparity processing stages leading to the formation of a proto-object representation upon which higher cognitive processes can act.


title: Neuron-derived Thioredoxin-80: a novel regulator of type-I interferon response in microglia

abstract: Oxidative stress and neuroinflammation play a central role in Alzheimers Disease (AD) pathogenesis. However, the mechanism by which these processes lead to neurodegeneration is still not fully understood. Thioredoxin-1 (Trx1) is an antioxidant protein that can be cleaved into a peptide known as Thioredoxin-80 (Trx80), which modulates monocyte function in the periphery and shows anti-amyloidogenic properties in the brain. In this study we aimed to further clarify the biological function of this peptide and its regulation in the brain. We show that neurons are the main producers of Trx80 in the brain. Trx80 levels increase in vivo both in normal aging and in young APPNL-G-F mouse model of amyloid pathology. Trx80 levels were increased in neurons in primary culture treated with either rotenone or 27-hydroxycholesterol, what suggests that Trx80 production is stimulated upon oxidative stress. RNA-sequencing followed by differential gene expression analysis revealed that Trx80 induces microglia activation into a phenotype compatible with interferon response microglia. Finally, we determine that the induction of this microglia phenotype by Trx80 is Trem2-dependent. This study identifies Trx80 as a novel neuron-derived signaling mechanism that modulates microglia function under stress conditions. Strategies to regulate Trx80 levels could be beneficial against AD pathology.


title: Choroid plexus tissue perfusion and secretory function in rats measured by non-invasive MRI reveal significant effects of anesthesia

abstract: The choroid plexus (ChP) of the cerebral ventricles is a source of cerebrospinal fluid (CSF) production and also plays a key role in immune surveillance at the level of blood-to-CSF-barrier (BCSFB). In this study, we quantify ChP blood perfusion and BCSFB mediated water exchange from arterial blood into ventricular CSF using non-invasive continuous arterial spin labelling magnetic resonance imaging (CASL-MRI). Systemic administration of anti-diuretic hormone (vasopressin) was used to validate BCSFB mediated water exchange as a metric of choroidal CSF secretory function. To further investigate the coupling between ChP perfusion and CSF secretory function, we characterized the effects of two anesthetic regimens known to have large-scale differential effects on cerebral blood flow. For quantification of ChP blood perfusion a multi-compartment perfusion model was employed, and we discovered that partial volume correction improved measurement accuracy. Vasopressin significantly reduced both ChP blood perfusion and CSF secretory function. ChP perfusion was significantly higher with pure isoflurane anesthesia (2-2.5%) when compared to a balanced anesthesia with dexmedetomidine and low-dose isoflurane (1.0 %), however there was no significant difference in BCSFB water secretory function. In summary, here we introduce a non-invasive, robust, and spatially resolved in vivo imaging platform to quantify ChP perfusion as well as BCSFB water secretory function which can be applied to study coupling of these two key parameters in future clinical translational studies.


title: Single-cell transcriptome profiles of Drosophila fruitless-expressing neurons from both sexes

abstract: Drosophila melanogaster reproductive behaviors are orchestrated by fruitless neurons. We performed single-cell RNA-sequencing on fru P1 pupal neurons. Uniform Manifold Approximation and Projection (UMAP) clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the UMAP clusters have sex-differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, we annotate clusters as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that express doublesex. Enriched marker gene analyses also shows that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters. In vivo spatial gene-expression links to the UMAP clusters are provided. A functional analysis of fru P1 circadian neurons shows they have dimorphic roles in sleep/activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes have fru P1 neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors.


title: The lateral intraparietal area preferentially supports stimulus selection in directed tasks compared to undirected free behavior

abstract: Our reactions to the sensory world depend on context. For instance, explicit directions to search for a particular object or feature will induce a different treatment of the sensory world than exploration without a fixed goal. To understand how we navigate the sensory world, it is necessary to understand how both directed search and undirected exploration are produced by the brain. The lateral intraparietal area (LIP) in the posterior parietal cortex has an established role in visual stimulus selection. However, most studies of LIPs role in stimulus selection focus primarily on highly trained, directed tasks, in which animals are given explicit cues as to which stimulus they should select. Here, we compare neural activity in LIP across two tasks. In one task, the animal is given an explicit direction to select one of two natural images from an array; in the other, the animal is allowed to choose an image freely based on their innate preferences. We find that LIP reliably encodes the eye movement prior to its execution only in the directed task, while the eye movement encoding in the undirected task emerges significantly later. Further, LIPs encoding of the images behavioral relevance emerges after the decision in both tasks. These results indicate that LIP preferentially supports stimulus selection in highly trained and directed behaviors, as opposed to free behavior.


title: Ambient RNA analysis reveals misinterpreted and masked cell types in brain single-nuclei datasets

abstract: Ambient RNA contamination in single-cell RNA sequencing (RNA-seq) is a significant problem, but its consequences are poorly understood. Here, we show that ambient RNAs in brain single-nuclei RNA-seq can have a nuclear or extra-nuclear origin with distinct gene set signatures. Both ambient RNA signatures are predominantly neuronal and we find that some previously annotated neuronal cell types are distinguished by ambient RNA. Strikingly, we also detect pervasive neuronal ambient RNA contamination in all glial cell types unless glia and neurons are physically separated prior to sequencing. We demonstrate that this contamination can be removed in silico. We also show that previous annotations of immature oligodendrocytes are likely glial cells contaminated with ambient RNAs. After ambient RNA removal, we detect extremely rare, committed oligodendrocyte progenitor cells, which were infrequently annotated in previous adult human brain datasets. Together, these results provide an in-depth analysis of ambient RNA contamination in brain single-cell datasets.


title: The Musashi proteins are post-transcriptional activators of protein expression and alternative exon splicing in vertebrate photoreceptors

abstract: The Musashi proteins, MSI1 and MSI2, are conserved RNA binding proteins with a role in the maintenance and renewal of stem cells. Contrasting with this role, retina and terminally differentiated photoreceptor cells express high levels of MSI1 and MSI2, indicating that the two proteins have a role unrelated to maintaining undifferentiated cell state. Here we show that the Musashi proteins are essential in mature photoreceptors. Combined knockout of Msi1 and Msi2 lead to loss of the retina response to light and progressive photoreceptor cell death. The two proteins are fully redundant as individual deletion of Msi1 or Msi2 did not produce a phenotype. To define the molecular functions underlying the requirement for Musashi in photoreceptors, we delineated their RNA targets and analyzed the effect of the combined Msi1/Msi2 knockout on transcript levels, pre-mRNA splicing, and protein expressions. We show distinct nuclear and cytoplasmic functions for the Musashi proteins in photoreceptor cells. In the nucleus, Musashi binding to the downstream proximal intron promotes splicing of alternative exons. Surprisingly, four conserved photoreceptor-specific alternative exons in genes critical for vision proved to be dispensable, leaving open questions about the selective pressures that lead to the conservation of these exons and the contribution of alternative splicing to the phenotype of the Musashi knockout. In the photoreceptor cell cytoplasm MSI1 and MSI2 act as activators of protein expression. The combined knockout of Msi1 and Msi2 reduced the levels of multiple proteins including proteins required for vision and photoreceptor survival.


title: The vigor paradox: saccade velocity during deliberation encodes utility of effortful actions

abstract:
During deliberation, as the brain considers its options, the neural activity representing the goodness of each option rises toward a threshold, and the choice is often dictated by the option for which the rise is fastest. Here we report a surprising correlate of these activities: saccade vigor. We engaged human subjects in a decision-making task in which they considered effortful options, each requiring walking various durations and inclines. As they deliberated, they made saccades between the symbolic representations of those options. These saccades had no bearing on the effort that they would later expend, yet as they deliberated, saccade velocities increased. The rate of rise in vigor was faster for saccades toward the option that they later indicated as their choice, and encoded the difference in the subjective value of the two effortful options. Once deliberation ended, following a brief delay the subjects indicated their choice by making another saccade. Remarkably, vigor for this saccade dropped to baseline and no longer encoded subjective value. These results are consistent with an urgency model of decision-making in which a global signal in the brain drives both the neural circuits that make decisions, and the neural circuits that make movements. Paradoxically, this common drive is shared between the oculomotor circuits and the decision-making circuits, even when the decision involves effortful expenditure during a future event.

SignificanceThere is a link between the decisions we make and the movements that follow. Not only do we prefer options of greater value, but we also move faster to acquire them. When deliberating between options, neural activity rises to a threshold and the option that wins this race is the one chosen. We report a potential correlate of this in the motor control circuits; during deliberation, saccade vigor to both options rise, but faster for the option ultimately chosen. Thus, our movements appear to mirror the neural activity conducting the decision-making process. Paradoxically, this is true even when the movements have no direct bearing on the decision at hand.


title: NLGC: Network Localized Granger Causality with Application to MEG Directional Functional Connectivity Analysis

abstract: Identifying the causal relationships that underlie networked activity between different cortical areas is critical for understanding the neural mechanisms behind sensory processing. Granger causality (GC) is widely used for this purpose in functional magnetic resonance imaging analysis, but there the temporal resolution is low, making it difficult to capture the millisecond-scale interactions underlying sensory processing. Magnetoencephalography (MEG) has millisecond resolution, but only provides low-dimensional sensor-level linear mixtures of neural sources, which makes GC inference challenging. Conventional methods proceed in two stages: First, cortical sources are estimated from MEG using a source localization technique, followed by GC inference among the estimated sources. However, the spatiotemporal biases in estimating sources propagate into the subsequent GC analysis stage, resulting in both false alarms and missing true GC links. Here, we introduce the Network Localized Granger Causality (NLGC) inference paradigm, which models the source dynamics as latent sparse multivariate autoregressive processes and estimates their parameters directly from the MEG measurements, without intermediate source localization, and employs the resulting parameter estimates to produce a precise statistical characterization of the detected GC links. We offer several theoretical and algorithmic innovations within NLGC and further examine its utility via comprehensive simulations and application to MEG data from an auditory task involving tone processing from both younger and older participants. Our simulation studies reveal that NLGC is markedly robust with respect to model mismatch, network size, and low signal-to-noise ratio, whereas the conventional two-stage methods result in high false alarms and mis-detections. We also demonstrate the advantages of NLGC in revealing the cortical network-level characterization of neural activity during tone processing and resting state by delineating task- and age-related connectivity changes.


title: Innate immune response and distinct metabolomic signatures together drive and shape the SARS-CoV-2-specific T cell response during COVID-19

abstract:
The underlying factors contributing to the evolution of SARS-CoV-2-specific T cell responses during COVID-19 infection remain unidentified. To address this, we characterized innate and adaptive immune responses with metabolomic profiling longitudinally at three different time points (0-3, 7-9, and 14-16 days post-COVID-19 positivity) from young mildly symptomatic active COVID-19 patients infected with the ancestral virus in mid-2020. We observed that anti-RBD IgG and viral neutralization are significantly reduced against the Delta variant compared to the ancestral strain. In contrast, compared to the ancestral strain, T cell responses remain preserved against the delta and omicron variants. We determined innate immune responses during the early stage of active infection in response to TLR 3/7/8 mediated activation in PBMCs and serum metabolomic profiling. Correlation analysis indicated PBMCs-derived proinflammatory cytokines, IL-6, IL-1{beta}, and IL-23, and the abundance of plasma metabolites, pyroglutamate, itaconate, and azelaic acid, were predictive of a robust SARS-CoV-2-specific Th1 response at a later stage (two weeks after PCR positivity). These observations may contribute to designing effective vaccines and adjuvants that promote innate immune responses and metabolites to induce long-lasting anti-SARS-CoV-2 specific T cells response.

Key MessageO_LIEarly proinflammatory innate cytokine response is critical for a robust T cell response during COVID-19.
C_LIO_LIWe report a set of plasma metabolites that significantly correlate with the innate and adaptive immune responses.
C_LI

Graphical Abstract

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category: immunology


title: Proinflammatory innate cytokines and metabolomic signatures predict T cell response in active COVID-19


title: Characterization of Morreton Virus (MORV) as a Novel Oncolytic Virotherapy Platform for Liver Cancers

abstract: Morreton virus (MORV) is a novel oncolytic Vesiculovirus, genetically distinct from vesicular stomatitis virus (VSV). we report that MORV induced potent cytopathic effects in a panel of cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines. In addition, high intranasal doses of MORV were not associated with significant adverse effects and were well tolerated in mice bearing liver tumor xenografts and syngeneic liver cancers. Furthermore, single intratumoral treatments with MORV (1 x 107 TCID50) triggered a robust antitumor immune response leading to substantial tumor regression and disease control in a syngeneic CCA model, using 10-fold lower dose compared to VSV (1 x 108 TCID50). In addition, MORV and VSV both induced prominent tumor growth delay in immunodeficient mice bearing Hep3B hepatocellular carcinoma (HCC) but not in mice bearing HuCCT-1 CCA xenografts. Our findings indicate that wild-type MORV is safe and can induce potent tumor regression in HCC and CCA animal models without adverse events via immune-mediated and immune-independent mechanisms. Further development and clinical translation of MORV as virotherapy for liver cancers are warranted.


title: HOIL1 regulates group 2 innate lymphoid cell numbers and type 2 inflammation in the small intestine

abstract: Patients with mutations in HOIL1 experience a complex immune disorder including intestinal inflammation. To investigate the role of HOIL1 in regulating intestinal inflammation, we employed a mouse model of HOIL1 deficiency. The ileum of Hoil1-/- mice displayed features of type 2 inflammation including tuft cell and goblet cell hyperplasia, and elevated expression of Il13, Il5 and Il25 mRNA. Inflammation persisted in the absence of T and B cells, and bone marrow chimeric mice revealed a requirement for HOIL1 expression in radiation-resistant cells to regulate inflammation. Although disruption of IL-4 receptor alpha (IL4R) signaling on intestinal epithelial cells ameliorated tuft and goblet cell hyperplasia, expression of Il5 and Il13 mRNA remained elevated. KLRG1hi CD90lo group 2 innate lymphoid cell (ILC2) were increased independent of IL4R signaling, tuft cell hyperplasia and IL-25 induction. Antibiotic treatment dampened intestinal inflammation indicating commensal microbes as a contributing factor. We have identified a key role for HOIL1, a component of the Linear Ubiquitin Chain Assembly Complex, in regulating type 2 inflammation in the small intestine. Understanding the mechanism by which HOIL1 regulates type 2 inflammation will advance our understanding of intestinal homeostasis and inflammatory disorders and may lead to the identification of new targets for treatment.


title: Increased receptor affinity and reduced recognition by specific antibodies contribute to immune escape of SARS-CoV-2 variant Omicron

abstract: In this report, we mechanistically reveal how the Variant of Concern (VOC) SARS-CoV-2 Omicron (B.1.1.529) escapes neutralizing antibody responses, by characterization of this variant, and wildtype Wuhan and Delta variant (B.1.617.2). Convalescent sera as well as sera obtained from participants who received two or three doses of mRNA vaccines (Moderna-mRNA-1273(R) or Pfizer-BNT162b2(R)) were used for comparison in this study. Our data demonstrate that both the Delta as well as Omicron variants exhibit higher affinity for the receptor ACE2, facilitating infection and causing antibody escape by receptor affinity (affinity escape), due to reduced ability of antibodies to compete with RBD-receptor interaction and virus neutralization. In contrast, only Omicron but not Delta variant escaped antibody recognition, most likely because only Omicron exhibit the mutation at position E484 associated with reduced recognition, resulting in further reduced neutralization (specificity escape). Nevertheless, the immunizations with RNA based vaccines resulted in marked viral neutralization in vitro for all strains, compatible with the fact that Omicron is still largely susceptible to vaccination-induced antibodies, despite affinity- and specificity escape.


title: A core transcriptional response for biofilm formation by Y. pseudotuberculosis

abstract: Previous transcriptional profiling of the enteropathogen Yersinia pseudotuberculosis during persistent stages of colonisation of mouse cecal lymphoid follicles indicated the possible involvement of biofilm in infection maintenance. Not much is known about the mechanisms responsible for biofilm formation by this pathogen, and most current knowledge is based on results of experiments conducted using the related Y. pestis pathogen that forms biofilm in the flea gut. In this study, we performed transcriptional profiling of Y. pseudotuberculosis in biofilms from different biofilm-inducing conditions, bile exposure, amino acid deprivation and in vivo mimicking conditions with and without oxygen. The comparison of differential expression of genes in biofilm versus planktonic bacteria showed a set of 54 core genes that were similarly regulated, independent of inducing condition. This set included many genes that were previously shown to be associated with biofilms, such as hutG, hsmF, hmsT and cpxP that were upreg-ulated and other genes such as hmsP and rfaH that were downregulated. There were also novel biofilm-associated genes, including genes encoding hypothetical proteins. To identify the genes involved in inducing biofilm formation, the gene expression of bacteria during an early initial phase when biofilm starts to form after induction by bile or amino acid depletion was determined. Comparisons of the resulting gene expression profiles with the profiles of non-induced bacteria incubated for the same period of time showed a set of core genes associated with early biofilm formation. This set included genes involved in quorum sensing, pili biogenesis and genes indicative of a potential metabolic shift involving nitrogen utilisation. Genes encoding components of sugar phosphotransferase systems were also up-regulated during biofilm induction. Assays of biofilm formation by bacteria deleted of some of these core genes showed that strains lacking hpr and luxS, which are known to be important for functional sugar phosphotransferase systems and quorum sensing, as well as glnL encoding a sensory histidine kinase were most negatively affected. Most of the deletion mutant strains tested were affected, but the effect was less severe, suggesting high levels of redundancy in the pathways involved in biofilm formation by this pathogen.

category: microbiology


title: Development of a tractable model system to mimic wood-ageing of beer on a lab scale

abstract: Wood-ageing of conventionally fermented beers is gaining increased attention in the production of sour beers with a noteworthy balance between sourness, wood aroma and flavour complexity. Besides the extraction of wood-derived compounds into the beer, wood-aged sours owe their layered flavour profile to the activity of a variety of  wild microorganisms that reside in the barrels or that emerge from the brewing or maturation environment. However, until now wood-ageing of craft beers largely remains a process of trial and error that often generates unexpected or undesirable results. Therefore, to better understand the process and develop control strategies to improve the consistency, predictability and overall quality of the resulting beer, more insight is needed into the interactions between the wood, the microorganisms and the maturing beer. Nevertheless, as studying these interactions on an industrial scale is highly challenging, the objective of this study was to develop a reproducible and easy-to-manipulate experimentally tractable system that can be used to study wood-ageing of beer on a lab scale. Barrel-ageing was mimicked in a 0.5 liter glass jar filled with beer and closed off by a wooden disk. Furthermore, the system was equipped with a synthetic community composed of four bacterial species (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevis and Pediococcus damnosus) and four fungal species (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciens and Saccharomyces cerevisiae) that represented key microbes previously identified in wood-ageing experiments with 225-liter barrels. In order to test the hypothesis that the barrel-ageing process of beer can be replicated in the simplified in-vitro system, the system was subjected to 60 days of ageing and microbial community dynamics and beer chemistry were compared with a 38-week industrial barrel-ageing experiment using the same beer. Beer samples were collected at regular time points and subjected to both qPCR assays targeting the eight selected species and chemical analysis. Results revealed that in vitro ageing showed similar trends in the temporal dynamics of the microbial populations and beer chemistry as those observed during 38-weeks of barrel-ageing in 225-liter barrels. Furthermore, results were found to be highly reproducible. Altogether, the in-vitro system was found to be a robust and reproducible system that has great potential to perform more in-depth research about the intricate interactions between microbes, wood and maturing beer and to develop control strategies to improve the consistency, predictability and overall quality of the resulting beer.


title: Phytochemical-induced mucin accumulation in the gastrointestinal lumen is independent of the microbiota

abstract: The mucus layer is critical to gastrointestinal health and ecology. Dietary phytochemicals are well documented to stimulate mucus production and secretion, but the underlying mechanism and effects on gut health are poorly understood. We fed germ-free and conventional mice diets containing approximately 0.4% of polyphenols per gram to determine if the phytochemical-induced accumulation of mucin in the gastrointestinal lumen is dependent on the microbiota. In addition, we assess how increased mucin shapes microbial communities in conventional mice. Germ-free mice receiving a pea (Pisum sativuum) seed coat proanthocyanidin-containing diet (PA) had greater levels of fecal mucin compared to the non-proanthocyanidin-containing (NPA) pea seed coat diet control (P < 0.05), confirming that fecal mucin accumulation is independent of the gut microbiota. Conventional mice fed the PA diet and a red osier dogwood (ROD; Cornus sericea) extract diet (DW) had higher mucin levels compared to a control diet without phytochemicals (P < 0.01 and P < 0.05, respectively). The increase in luminal mucin was associated with consistent increases in bacterial taxa belonging to Lachnospiraceae and [Clostridium] leptum species and a decrease in Romboutsia species. We conclude that phytochemicals have the ability to alter gut microbial ecology by increasing the amount of mucin in the gastrointestinal lumen.


title: Bartonella taylorii: A model organism for studying Bartonella infection in vitro and in vivo

abstract: Bartonella spp. are Gram-negative facultative intracellular pathogens that infect diverse mammals and cause a long-lasting intra-erythrocytic bacteremia in their natural host. These bacteria translocate Bartonella effector proteins (Beps) into host cells via their VirB/VirD4 type 4 secretion system (T4SS) in order to subvert host cellular functions, thereby leading to the downregulation of innate immune responses. Most studies on the functional analysis of the VirB/VirD4 T4SS and the Beps were performed with the major zoonotic pathogen Bartonella henselae for which efficient in vitro infection protocols have been established. However, its natural host, the cat, is unsuitable as an experimental infection model. In vivo studies were mostly confined to rodent models using rodent-specific Bartonella species, while the in vitro infection protocols devised for B. henselae are not transferable for those pathogens. The disparities of in vitro and in vivo studies in different species has hampered progress in our understanding of Bartonella pathogenesis. Here we describe the murine-specific strain B. taylorii IBS296 as a new model organism facilitating the study of bacterial pathogenesis both in vitro in cell cultures and in vivo in laboratory mice. We implemented the split NanoLuc luciferase-based translocation assay to study BepD translocation through the VirB/VirD4 T4SS. We found increased effector-translocation into host cells if the bacteria were grown on tryptic soy agar (TSA) plates and experienced a temperature shift immediately before infection. The improved infectivity in vitro could be correlated to an upregulation of the VirB/VirD4 T4SS. Using our adapted infection protocols, we showed BepD-dependent immunomodulatory phenotypes in vitro. In mice, the implemented growth conditions enabled infection by a massively reduced inoculum without having an impact on the course of the intra-erythrocytic bacteremia. The established model opens new avenues to study the role of the VirB/VirD4 T4SS and the translocated Bep effectors in vitro and in vivo.


title: Kaposi's sarcoma-associated herpesvirus induces specialised ribosomes to efficiently translate viral lytic mRNAs

abstract: Historically, ribosomes have been viewed as unchanged homogeneous macromolecular machines with no intrinsic regulatory capacity for mRNA translation. However, an emerging concept is that heterogeneity of ribosomal composition exists, which can exert a regulatory function or specificity in translational control. This is supported by recent discoveries identifying compositionally distinct  specialised ribosomes that actively regulate mRNA translation. Viruses lack their own translational machinery and impose a high translational demand on the host cell during replication. Here we explore the possibility that Kaposis sarcoma-associated herpesvirus (KSHV) can manipulate host ribosome biogenesis during infection to produce specialised ribosomes which preferentially translate viral transcripts. Quantitative proteomic analysis has identified changes in the stoichiometry and composition of precursor ribosomal complexes during the switch from latent to lytic KSHV replication. Intriguingly, we demonstrate the enhanced association of ribosomal biogenesis factors BUD23 and NOC4L, and a previously uncharacterised KSHV lytic protein, ORF11, with small ribosomal subunit precursor complexes during lytic KSHV infection. Notably, BUD23 depletion resulted in significantly reduced viral gene expression and progression through the lytic cascade, culminating in a dramatic reduction of infectious virion production. Importantly, ribosome profiling demonstrated that BUD23 is essential for the reduced association of ribosomes with KSHV uORFs in late lytic genes, required for the efficient translation of the main open reading frame. Together our results provide new mechanistic insights into KSHV-mediated manipulation of cellular ribosome composition inducing a population of specialised ribosomes to facilitate efficient translation of viral mRNAs.


title: Blocking unwanted sequences by target specific high-affinity binding oligonucleotides during transcriptome library preparation

abstract: RNA sequencing has become the gold standard for transcriptome analysis but comes with an inherent limitation with respect to quantification of low abundant transcripts. In contrast to microarray technology, RNA-sequencing reads are proportionally divided across transcripts. Therefore, low abundant RNAs can be out-competed by highly abundant--and sometimes non-informative--RNA species. We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR-amplification of specific RNA transcripts, hereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different RNA molecules and library preparation strategies, including YRNAs in small RNA-sequencing of human plasma samples, mitochondrial rRNAs in both 3 end sequencing and long-read sequencing, and MALAT1 in single-cell 3 end sequencing. We demonstrate that the blocking strategy is extremely efficient, reproducible and specific, and generally results in better transcriptome coverage and complexity. Our method does not require modifications of the library preparation procedure apart from adding blocking oligonucleotides to the RT reaction and can thus be easily integrated in virtually any RNA sequencing library preparation protocol.

category: molecular biology


title: SELF PRUNING 3C is a flowering repressor that modulates seed germination, root architecture and drought responses

abstract:
Allelic variation in the CETS (CENTRORADIALIS, TERMINAL FLOWER 1, SELF PRUNING) gene family has been shown to control agronomically important traits in many crops. CETS genes encode phosphatidylethanolamine binding proteins (PEBPs) that have a central role in flowering time control as florigenic and anti-florigenic signals. The great expansion of CETS genes in many species suggests that the functions of this family go beyond flowering. Here, we characterize the tomato SELF PRUNING 3C (SP3C) gene, and show that besides acting as a flowering repressor it also regulates seed germination and modulates root architecture. We show that loss of SP3C function in CRISPR/Cas9-generated mutant lines accelerates seed germination and increases root length with lower root side branching. Higher SP3C expression in transgenic lines promotes the opposite effects and also improves tolerance to water stress in seedlings. These discoveries provide insights into the role of SP paralogs in agronomically relevant traits and support future exploration of the involvement of CETS genes in abiotic stress responses.

HighlightThe SELF PRUNING 3C (SP3C) gene is a repressor of flowering in tomato and exhibits additional functions, acting as a repressor of seed germination and modulating root architecture.


title: A critical role for heme synthesis and succinate in the regulation of pluripotent states transitions

abstract: Using embryonic stem cells (ESCs) in regenerative medicine or in disease modeling requires a complete understanding of these cells. Two main distinct developmental states of ESCs have been stabilized in vitro, a naive pre-implantation stage and a primed post-implantation stage. Based on two recently published CRISPR-Cas9 knockout functional screens, we show here that the exit of the naive state is impaired upon heme biosynthesis pathway blockade, linked to the incapacity to activate MAPK- and TGF{beta}-dependent signaling pathways. In addition, heme synthesis inhibition promotes the acquisition of 2 cell-like cells in a heme-independent manner caused by a mitochondrial succinate accumulation and leakage out of the cell. We further demonstrate that extra-cellular succinate acts as a paracrine/autocrine signal, able to trigger the 2C-like reprogramming through the activation of its plasma membrane receptor, SUCNR1. Overall, this study unveils a new mechanism underlying the maintenance of pluripotency under the control of heme synthesis.

category: developmental biology


title: Thymus formation in uncharted embryonic territories.

abstract:
The thymus is a conserved organ among vertebrates, derived from the endoderm of distinct pharyngeal pouches (PP), whose location and number vary in different species. Together with reports of sporadic ectopic thymus locations in mice and humans, this suggests that the potential to make a thymus resides in a broader region of the PP endoderm than previously ascribed.

Using the chick-quail chimera system, we explore this hypothesis and test the capacity of non-canonical pouches to participate in thymus formation. We further ask if the local mesenchyme of pharyngeal arches (PA) could also play a role in the regulation of thymus formation.

After testing several embryonic tissue associations, we mapped the pharyngeal endoderm regions with thymus potential to the second and third/fourth pharyngeal pouches (2PP and 3/4PP). We further identified mesenchyme regions that regulate this potential to the the 3/4 pharyngeal arches and to the dorsal region of the second arch, with positive and negative influences, respectively.

Transcriptomic analysis of these tissues helped us to delineate a common genetic program correlated with thymus potential in the endoderm, and to define distinct signalling pathways acting in mesenchymal environments to help patterning pharyngeal arches. Together, these results provide new information about the initial specification of thymus primordia in the embryo and might help in improving thymus organoid systems.


title: Continuing decline of the eastern quoll in Tasmania

abstract: Like many other Australian mammals, the eastern quoll (Dasyurus viverrinus) was widespread on the Australian mainland but went extinct there during the 20th century. The species remained abundant in Tasmania until a rapid decline occurred from 2001 to 2003, coinciding with a period of unsuitable weather. We provide an updated analysis of eastern quoll population trends in Tasmania by analysing a Tasmania-wide time series of annual spotlight counts (1985-2019). Eastern quolls were widespread and abundant in Tasmania until the early 2000s. A distinct change occurred in the early 2000s in the east and northeast, which led to severe population reductions. However, we present new evidence of an earlier decline in the north (mid-1990s) and a more recent decline around 2009 in the south. Range-wide declines have continued unabated during the last decade, resulting in a [~]67% decline (since the late 1990s) in the area with high quoll abundance. Although the timing of the major decline in the early 2000s coincided with unfavourable weather, the continuing decline and more recent change points suggest other causes are also involved. We can no longer assume that the existence of eastern quolls in Tasmania ensures the species long-term survival, highlighting the urgent need to increase efforts to conserve the remaining populations in Tasmania.

category: ecology


title: MitoSearch: Construction of fish species composition database with environmental DNA using public databases

abstract: Existing fish species distribution databases have the following problems: the possibility of species identification errors due to plasticity of morphological characteristics, the impossibility of quantitative composition, and the difficulty of geographically and time-series integrated interpretation. To solve the above problems, we constructed MitoSearch database. It is a fish species distribution database using 2,160 samples of variable regions of 12S rRNA genes amplified by MiFish primers registered at NCBI/EBI/DDBJ, and visualizes the fish species composition data on a map. By integrating a large amount of data registered in public databases, MitoSearch was aimed to provide the comprehensive understanding of spatial and temporal distribution of organisms on a global scale.


title: A chronotype effect: circadian-period variation underlies the local adaptation of photoperiodism in the short-day plant Lemna aequinoctialis.

abstract: Phenotypic variation is the basis for trait adaptation via evolutionary selection.1, 2, 3 However, the driving forces behind the quantitative trait variations remain unclear owing to their complexity at the molecular level.4, 5, 6 This study focused on the natural variation of the free-running period (FRP) of the circadian clock because FRP is a determining factor of the internal clock phase (chronotype), which is responsible for physiological timing during a day.7 Although natural variations in FRP have been widely reported,8, 10, 11 few studies have shown the association between FRP and adaptive temporal traits. As a clock-dependent physiological process, photoperiodism is a typical target of local adaptation.12, 13 Lemna aequinoctialis in Japan is a paddy-field duckweed exhibiting a latitudinal cline of critical day-lengths (CDLs) for short-day flowering.14 To investigate the relationship between FRP and CDL, we collected 72 strains of L. aequinoctialis within a latitudinal range between 31.5{degrees}N to 43.8{degrees}N. We found a significant correlation (P = 7.5E-8) between FRPs and locally adaptive CDLs, confirming that the variation in FRP-dependent chronotypes underlies geographically differentiated photoperiodism. Diel transcriptome analysis revealed that the induction timing of a florigen gene is key for connecting chronotypes to photoperiodism at the molecular level. Based on these results, we propose a fundamental rule concerning the "chronotype effect" in evolution: the variation of FRP functions as a resource for the variation of temporal traits. This study highlights the adaptive significance of FRP variation and provides a reason for the maintenance of FRP variation in natural populations.


title: Circadian-period variation underlies the local adaptation of photoperiodism in the short-day plant Lemna aequinoctialis.


title: Cross-generational heritability analysis of physiological traits in Porites astreoides across an inshore-offshore gradient in the Lower Florida Keys

abstract: Estimating the heritable genetic variation in fitness-related traits is key to projecting the adaptive evolution of organisms in response to a changing environment. While heritability studies on reef-building corals to date support adaptive capacity, little is known about the dynamics of trait heritability across life stages in which distinct selective pressures can have long-lasting effects both within and across generations. In this study, we obtained heritability estimates for energetic and thermal stress response traits in larval, recruit, and adult Porites astreoides from two populations in the Lower Florida Keys. To induce bleaching phenotypes among individual families, larvae were exposed to a 4-day thermal stress at 32 {degrees}C, whereas adults and recruits received the same treatment for 22 days. Origin-dependent tolerance was observed in two life stages where offshore recruits lost more symbiont cells under heat than inshore recruits compared to their respective controls and heat-treated offshore adults suffered a greater loss in total protein content. Surprisingly, larvae appeared to be largely insensitive to heat regardless of origin. Broad sense heritability (H2) estimates varied greatly among traits and life stages, which may reflect changes in the relative importance of genetic and environmental variation throughout development. Notably, more than 80% of the variation in larval chlorophyll a concentration was attributed to genetic factors. The overall moderate to high H2 estimates measured here suggest these corals have considerable potential to adapt to environmental change.


title: Niche-based deterministic assembly processes strengthen the effects of β-diversity on metacommunity functioning of marine bacterioplankton

abstract: Studies at a local community () level have shown that biodiversity positively affects ecosystem functioning due to niche-based deterministic processes like resource partitioning. Extending to a metacommunity ({beta}) level, we hypothesize that {beta} diversity also positively affects metacommunity functioning. We further hypothesize that the {beta} diversity effect is stronger (more positive) when {beta} diversity is increased by deterministic/non-random assembly processes. To test the hypotheses, we collected bacterioplankton along a transect of 6 stations in the southern East China Sea in 14 cruises. All 6 stations within a cruise were regarded as a metacommunity. For any pairs of the 6 stations, the Bray-Curtis index and summed bacterial biomass were calculated to represent {beta} diversity and metacommunity functioning, respectively. We also calculated deviation of observed mean pairwise phylogenetic similarities among species from random to represent the deterministic influences of assembly processes. We found that bacterial {beta} diversity generally positively affects metacommunity functioning; however, the {beta} diversity effect varied among cruises. Cross-cruise comparison indicates that the {beta} diversity effect increased with the deterministic assembly processes selecting for phylogenetically dissimilar species. This study extends the biodiversity-ecosystem functioning research to a metacommunity level, complementing the current framework by considering community assembly in natural environments.


title: Focusing the GWAS Lens on days to flower using latent variable phenotypes derived from global multi-environment trials

abstract: Adaptation constraints within crop species have resulted in limited genetic diversity in some breeding programs and/or areas where new crops have been introduced, e.g., lentil (Lens culinaris Medik.) in North America. An improved understanding of the underlying genetics involved in phenology-related traits is valuable knowledge to aid breeders in overcoming limitations associated with unadapted germplasm and expanding their genetic diversity by introducing new, exotic material. We used a large, 18 site-year, multi-environment dataset, phenotyped for phenology-related traits across nine locations and over three years, along with accompanying latent variable phenotypes derived from a photothermal model and principal component analysis (PCA) of days from sowing to flower (DTF) data for a lentil diversity panel (324 accessions) which has also been genotyped with an exome capture array. Genomewide association studies (GWAS) on DTF across multiple environments helped confirm associations with known flowering time genes and identify new quantitative trait loci (QTL), which may contain previously unknown flowering time genes. Additionally, the use of latent variable phenotypes, which can incorporate environmental data such as temperature and photoperiod as both GWAS traits and as covariates, strengthened associations, revealed additional hidden associations, and alluded to potential roles of the associated QTL. Our approach can be replicated with other crop species, and the results from our GWAS serve as a resource for further exploration into the complex nature of phenology-related traits across the major growing environments for cultivated lentil.

category: genetics


title: Assembly and phylogeographic analysis of novel Taenia solium mitochondrial genomes reveal further differentiation between and within Asian and African-American genotypes

abstract:
BackgroundTaenia solium is a parasite that hampers human health, causing taeniasis and cysticercosis. The genetic variability in its mitochondrial genome is related to the geographical origin of the specimen. Two main genotypes have been identified: The Asian and the African-American. The geographic genetic variability is expected to cause different clinical manifestations. Thus, characterizing differences between and within genotypes is crucial for completing the epidemiology of T. solium diseases.

Methods/Principal FindingsHere, two Peruvian (one complete and one partial; 7,811X and 42X of coverage, respectively) and one Mexican (complete, 3,395X) T. solium mitochondrial genomes were assembled using the Chinese reference. Variant calling with respect to the reference was performed. Thirteen SNPs that involved a change in the amino acid physicochemical nature were identified. Those were present in all the assembled genomes and might be linked to differences in aerobic respiration efficiency between Latin American (African-American) and Asian genotypes. Then, phylogeographic studies were conducted using Cytochrome C oxidase subunit I and cytochrome B from these genomes and other isolates. The analysis showed that Indonesian samples are the most ancient and related to the modern T. solium ancestor of the Asian genotype. Finally, a consistent subdivision of the African-American genotype into two subgroups was found. One subgroup relates to East African countries, while the other is West Africa. The East African linage suggests a previously unnoticed influence of the Indian Ocean trade in the genetic structure of Latin America T. solium.

Conclusions/SignificanceOverall, this study reports novel mitochondrial genomes valuable for further studies. New Latin American SNPs were identified and suggest metabolic differences between parasites of the Asian and African-American genotypes. Moreover, the phylogeographic analysis revealed differences within each genotype that shed light on T. soliums historical spread. Overall, the results represent an important step in completing T. solium genetic epidemiology.

Author SummaryTaenia solium is a human parasite that causes taeniasis and cysticercosis. Eradicated from developed countries, they are still a public health problem in developing nations. T. solium differences in the mitochondrial genetic material depend on its geographical origin. This is expected to cause different clinical manifestations. Despite the importance of genetics to the epidemiology of T. solium diseases, few efforts have been made to assemble and compare their genomes. We aimed to help fill this knowledge gap by assembling three mitochondrial genomes from Latin America and comparing them to the Chinese reference. Additionally, two genes from the Latin American genomes and from other isolates were employed to assess T. solium genetic distribution. We found thirteen mutations with respect to the Chinese genome present in all Latin American samples, which involved a change in the amino acid physicochemical nature. Those might be causing metabolic differences between Asian and Latin American parasites that could change their affinity to specific human tissues. Moreover, we determined that Indonesian samples are the most ancient and related to the modern T. solium ancestor. Finally, we identified a previously unnoticed influence of East African countries in T. solium phylogeny, with which our assembled genomes are closely related.


title: Multi-omics comparison of malignant and normal uveal melanocytes reveals novel molecular features of uveal melanoma.

abstract: Uveal Melanoma (UM) is a rare cancer resulting from the transformation of melanocytes residing in the uveal tract. Integrative analysis has identified four molecular and clinical subsets in UM. To improve our understanding of UM we performed extensive multi-omics characterization comparing pure melanoma populations obtained from two aggressive UM patient-derived xenograft models with normal choroidal melanocytes. Our study addresses for the first time DNA optical mapping, specific histone mark modifications, DNA topology analysis by Hi-C. Gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. Our study also identified a recurrent deletion in the BAP1 promoter which results in the absence of expression of BAP1 which is associated with high risk of metastases in UM patients. Chromatin topology changes are associated with up-regulation of PRAME, a recognized independent prognostic biomarker in UM and potential therapeutic target. Our findings illustrate how multi-omics integrative approaches can improve the understanding of tumorigenesis and reveals two novel mechanisms of gene expression dysregulation in UM.

category: genomics


title: Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells

abstract: Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we established a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of [~]100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a new standard platform with which to pursue the design of noncoding regions for genome writing in human cells.

category: bioengineering


title: Fabrication of Conductive Hollow Microfibers for Encapsulation of Astrocyte Cells

abstract: The manufacturing of 3D cell scaffoldings provides advantages for modeling diseases and injuries by physiologically relevant platforms. A triple-flow microfluidic device was developed to rapidly fabricate alginate/graphene hollow microfibers based on the gelation of alginate induced with CaCl2. This five-channel pattern actualized continuous mild fabrication of hollow fibers under an optimized flowing rate ratio of 300: 200: 100 L.min-1. The polymer solution was 2.5% alginate in 0.1% graphene, and a 30% polyethylene glycol solution was used as the sheath and core solutions. The morphology and physical properties of microstructures were investigated by scanning electron microscopy, electrochemical, and surface area analyzers. Subsequently, these conductive microfibers biocompatibility was studied by encapsulating mouse astrocyte cells within these scaffolds. The cells could successfully survive both the manufacturing process and prolonged encapsulation for up to 8 days. These unique 3D hollow scaffolds could significantly enhance the available surface area for nutrient transport to the cells. In addition, these conductive hollow scaffolds illustrated unique advantages such as 0.728 cm3.gr-1 porosity and twice more electrical conductivity in comparison to alginate scaffolds. The results confirm the potential of these scaffolds as a microenvironment that supports cell growth.


title: A deep unsupervised language model for protein design

abstract: Protein design aims to build new proteins from scratch thereby holding the potential to tackle many environmental and biomedical problems. Recent progress in the field of natural language processing (NLP) has enabled the implementation of ever-growing language models capable of understanding and generating text with human-like capabilities. Given the many similarities between human languages and protein sequences, the use of NLP models offers itself for predictive tasks in protein research. Motivated by the evident success of generative Transformer-based language models such as the GPT-x series, we developed ProtGPT2, a language model trained on protein space that generates de novo protein sequences that follow the principles of natural ones. In particular, the generated proteins display amino acid propensities which resemble natural proteins. Disorder and secondary structure prediction indicate that 88% of ProtGPT2-generated proteins are globular, in line with natural sequences. Sensitive sequence searches in protein databases show that ProtGPT2 sequences are distantly related to natural ones, and similarity networks further demonstrate that ProtGPT2 is sampling unexplored regions of protein space. AlphaFold prediction of ProtGPT2-sequences yielded well-folded non-idealized structures with embodiments as well as large loops and revealed new topologies not captured in current structure databases. ProtGPT2 has learned to speak the protein language. It has the potential to generate de novo proteins in a high throughput fashion in a matter of seconds. The model is easy-to-use and freely available.


title: CRISPRcleanR WebApp: an interactive web application for processing genome-wide pooled CRISPR-Cas9 viability screen

abstract:
A major limitation of pooled CRISPR-Cas9 viability screens is the high false-positive rate in detecting essential genes arising from copy number-amplified (CNA) regions of the genome. To solve this issue, we developed CRISPRcleanR: a widely-used computational method implemented as R/python package and in a dockerized version. CRISPRcleanR detects and corrects biased gene-independent responses to CRISPR-Cas9 targeting in an unsupervised fashion, and it accurately reduces false-positive signals, while maintaining sensitivity in identifying relevant genetic dependencies. Here we present CRISPRcleanRWebApp, a web-based application that enables access to CRISPRcleanR through an intuitive graphical web interface. CRISPRcleanRWebApp removes the complexity of low-level R/python-language user interactions, including a range of interactively explorable plots. In addition, it supports a wider range of CRISPR guide RNAs libraries with respect to the original package. CRISPRcleanRWebApp is freely available and open to all users at: https://iorio-apps.fht.org/ccr.

HighlightsO_LICRISPR-Cas9 screens are widely used for the identification of cancer dependencies
C_LIO_LIFalse-positives in CRISPR-Cas9 screens arise from targeting copy-number (CN) amplified genomic regions
C_LIO_LICRISPRcleanR corrects this bias in an unsupervised fashion
C_LIO_LICRISPRcleanRWebApp is a web user-friendly front-end for CRISPRcleanR
C_LIO_LICRISPRcleanRWebApp supports a wider range of CRISPR libraries and supports interactive results explorations
C_LI

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org.highwire.dtl.DTLVardef@17dadb6org.highwire.dtl.DTLVardef@17b8b4org.highwire.dtl.DTLVardef@15f4c24org.highwire.dtl.DTLVardef@17fd030_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: Ion channel model reduction using manifold boundaries

abstract: Mathematical models of voltage-gated ion channels are used in basic research, industrial and clinical settings. These models range in complexity, but typically contain numerous variables representing the proportion of channels in a given state, and parameters describing the voltage-dependent rates of transition between states. An open problem is selecting the appropriate degree of complexity and structure for an ion channel model given data availability. Here, we simplify a model of the cardiac human Ether-a-go-go Related Gene (hERG) potassium ion channel, which carries cardiac IKr, using the manifold boundary approximation method (MBAM). The MBAM approximates high-dimensional model-output manifolds by reduced models describing their boundaries, resulting in models with fewer parameters (and often variables). We produced a series of models of reducing complexity starting from an established 5-state hERG model with 15 parameters. Models with up to 3 fewer states and 8 fewer parameters were shown to retain much of the predictive capability of the full model and were validated using experimental hERGla data collected in HEK293 cells at 37{degrees}C. The method provides a way to simplify complex models of ion channels that improves parameter identifiability and will aid in future model development.


title: Prokaryotic genome design is ruled by the mechanical properties and complexity of the DNA sequence

abstract: Although we represent the genetic code using four letters, namely A, T, G, and C, it is a chemical entity with sequence-dependent physical properties and a spatial extent. In other words, it can be said that genetic information is compressed and recorded in the genomic DNA as a storage medium with a spatial spread. This study analyzed prokaryotic genomes in terms of their physical properties and complexity. The findings clearly indicated that a mathematic equation can be used to express the relationship between the length of a genome and the upper limit of its rigidity. Furthermore, many prokaryotes have been shown to maximize DNA complexity under various physical constraints. Here, this relationship between rigidity and complexity was found to be shaped like an entropy function. It was also clarified that codon usage tendencies are affected by the physical properties and complexity of the DNA sequence. Moreover, the analysis of codons encoding two consecutive synonymous amino acids suggests that many prokaryotic codon combinations tend to increase genomic DNA complexity. This study revealed the macro-level rules for packing genomic DNA into prokaryotic cells.


title: Insight into Polyproline II Helical Bundle Stability and Folding from NMR Spectroscopic Characterization of the Snow Flea Antifreeze Protein Denatured State

abstract: The use of PPII helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. To get atomic level information on the conformational ensemble and entropy of the reduced denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at three experimental conditions. No significant populations of preferred secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation of the two disulfide bonds during nascent folding. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured /{beta} proteins both on fast ps/ns as well as slower s/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.


title: Cardiac ventricular myosin and slow skeletal myosin exhibit dissimilar chemo-mechanical properties despite the same myosin heavy chain isoform.

abstract: The myosin II motors are ATP-powered, force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta ({beta}-MyHC) is primarily expressed in the ventricular myocardium and slow-twitch muscle fibers, such as in M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular {beta}-cardiac myosin ({beta}M-II); however, the direct assessment of detailed biochemical and mechanical features of the native myosins is limited. By employing the optical trapping method, we examined the mechanochemical properties of the native myosins isolated from rabbit heart ventricle and M. soleus muscles at the single-molecule level. Contrary to previous reports, the purified motors from the two tissue sources, despite the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. {beta}M-II was [~]threefold faster in the actin filament-gliding assay than SolM-II. The maximum acto-myosin (AM) detachment rate derived in single-molecule assays was [~]threefold higher in {beta}M-II. The stroke size for both myosins was comparable. The stiffness of the  AM rigor cross-bridge was also similar for both the motor forms. The stiffness of {beta}M-II was found to be determined by the nucleotide state of the actin-bound myosin. Our analysis revealed distinct kinetic differences, i.e., a higher AM detachment rate for the {beta}M-II, corresponding to the ADP release rates from the cross-bridge, thus elucidating the observed differences in the motility driven by {beta}M-II and SolM-II. These studies have important implications for the future choice of tissue sources to gain insights into cardiomyopathies


title: Pan-cancer distribution of cleaved cell-surface Amphiregulin, the target of the GMF-1A3 antibody drug conjugate

abstract: Amphiregulin (AREG) is a transmembrane protein which, following TACE/ADAM17-dependent cleavage, releases a soluble Epidermal Growth Factor Receptor ligand domain that promotes proliferation of normal and malignant cells. Expression of Amphiregulin has been described by immunohistochemistry in several tumor types, including lung, prostate, head and neck, gastric, pancreatic and breast cancers but evidence for a functional requirement for Amphiregulin in these malignancies is more limited. We have previously described the development of a monoclonal antibody, GMF-1A3, that selectively recognizes the Amphiregulin epitope that is revealed following cleavage by TACE/ADAM17 and demonstrated that drug conjugates of this antibody have anti-tumor activity in mouse models. By directly evaluating Amphiregulin cleavage, immunohistochemistry on tissue specimens using this antibody can be used to evaluate the extent to which Amphiregulin is being proteolytically processed in cancer, which is a more direct measure of Amphiregulin activity. As a potential companion diagnostic for this antibody-drug conjugate, this immunohistochemistry assay allows identification of tumors with high levels of the cleaved Amphiregulin target. Here we evaluate levels of cleaved Amphiregulin in 370 specimens from 10 tumor types and demonstrate that it is widely expressed in solid tumors and is especially common (more than 50% of cases) in breast, prostate, liver and lung cancer.

category: cancer biology


title: Flotillin-2 regulates EGFR activation, degradation, and cancer growth

abstract: Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.


title: Structure and function of diadenylate cyclase DacM from Mycoplasma ovipneumoniae

abstract: Cyclic diadenosine monophosphate (c-di-AMP) is a second-messenger nucleotide that is produced by many bacteria. C-di-AMP can not only regulate bacterial growth, cell-wall homeostasis, ion transport and gene transcription, but can also be recognized by multiple sensor / receptor proteins in infected host cells to trigger an innate immune response. Mycoplasma ovipneumoniae causes non-progressive pneumonia in both sheep and goats. Here, we analyzed c-di-AMP signaling in M. ovipneumoniae, which is a genome-reduced obligately pathogenic bacterium. Our results demonstrate that these bacteria can produce c-di-AMP, and we could identify the diadenylate cyclase, which was named DacM. The enzyme was found to utilize both ATP and ADP to synthesize c-di-AMP, resembling CdaM from a novel family of diadenylate cyclases first found in Mycoplasma pneumoniae. Furthermore, we present the crystal structures of DacM in the apo state and substrate-bound state at 3 [A] and 1.9 [A] resolution, respectively. Mutation of residues Asp112, Gly113, Tyr128, Phe129, and Arg143 surrounding the active sites to Ala were lethal to DacM enzymatic activity. These structures provide valuable insights into the biochemistry of c-di-AMP, and offer a basis for the structure-based design of new drugs for animal husbandry.

category: pathology


title: Adipocyte-specific ablation of the Ca2+ pump SERCA2 impairs whole-body metabolic function and reveals the diverse metabolic flexibility of white and brown adipose tissue.

abstract:
ObjectiveSarco/endoplasmic reticulum Ca2+-ATPase (SERCA) transports Ca2+ from the cytosol into the ER and is essential for appropriate regulation of intracellular Ca2+ homeostasis. The objective of this study was to test the hypothesis that SERCA pumps are involved in the regulation of white adipocyte hormone secretion and other aspects of adipose tissue function and that this control is disturbed in obesity-induced type-2 diabetes.

MethodsSERCA expression was measured in isolated human and mouse adipocytes as well as in whole mouse adipose tissue by Western blot and RT-qPCR. To test the significance of SERCA2 in adipocyte functionality and whole-body metabolism, we generated adipocyte-specific SERCA2 knockout mice. The mice were metabolically phenotyped by glucose tolerance and tracer studies, histological analyses, measurements of glucose- stimulated insulin release in isolated islets, and gene/protein expression analyses. We also tested the effect of pharmacological SERCA inhibition and genetic SERCA2 ablation in cultured adipocytes. Intracellular and mitochondrial Ca2+ levels were recorded with dual-wavelength ratio imaging and mitochondrial function was assessed by Seahorse technology.

ResultsWe demonstrate that SERCA2 is downregulated in white adipocytes from patients with obesity and type-2 diabetes as well as in adipocytes from diet-induced obese mice. SERCA2-ablated adipocytes display disturbed Ca2+ homeostasis associated with upregulated ER stress markers and impaired hormone release. These adipocyte alterations are linked to mild lipodystrophy, reduced adiponectin levels, and impaired glucose tolerance. Interestingly, adipocyte-specific SERCA2 ablation leads to increased glucose uptake in white adipose tissue while glucose uptake is reduced in brown adipose tissue. This dichotomous effect on glucose uptake is due to differently regulated mitochondrial function. In white adipocytes, SERCA2 deficiency triggers an adaptive increase in Fgf21, increased mitochondrial UCP1 levels, and increased oxygen consumption rate (OCR). In contrast, brown SERCA2 null adipocytes display reduced OCR despite increased mitochondrial content and UCP1 levels compared to wild type controls.

ConclusionsOur data suggest causal links between reduced white adipocyte SERCA2 levels, deranged adipocyte Ca2+ homeostasis, adipose tissue dysfunction and type-2 diabetes.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/483886v1_ufig1.gif" ALT="Figure 1">
View larger version (23K):
org.highwire.dtl.DTLVardef@ce9737org.highwire.dtl.DTLVardef@129abaeorg.highwire.dtl.DTLVardef@c63342org.highwire.dtl.DTLVardef@4f47fe_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIAdipocyte SERCA2 is downregulated in human subjects with type-2 diabetes
C_LIO_LILoss of SERCA2 disturbs the intracellular Ca2+ homeostasis in adipocytes
C_LIO_LIImpaired metabolism and altered adipokine levels in adipocyte-SERCA2 null mice
C_LIO_LILoss of SERCA2 accelerates metabolic processes in white adipocytes
C_LIO_LILoss of SERCA2 impairs the mitochondrial function of brown adipocytes
C_LI

category: physiology


title: Odors drive feeding through gustatory receptor neurons in Drosophila

abstract: Odors are intimately tied to the taste system to aid food selection and determine the sensory experience of food. However, how smell and taste are integrated in the nervous system to drive feeding behavior remains largely unknown. We show in Drosophila that odors alone activate gustatory receptor neurons (GRNs) and trigger proboscis extension reflex (PER), a canonical taste-evoked feeding behavior. Odor-evoked PER requires the function of sugar-sensing GRNs but not the olfactory organs. Calcium imaging shows that GRNs directly respond to odors. Odor-evoked PER is mediated by the Gr5a receptor, and is bidirectionally modulated by specific olfactory binding proteins. Finally, odors and sucrose co-applied to GRNs synergistically enhance PER. These results reveal a cell-intrinsic mechanism for odor-taste multimodal integration that takes place as early as in GRNs, indicating that unified chemosensory experience is a product of layered integration in peripheral neurons and in the brain.


title: BLMM: Parallelised Computing for Big Linear Mixed Models

abstract: Within neuroimaging large-scale, shared datasets are becoming increasingly commonplace, challenging existing tools both in terms of overall scale and complexity of the study designs. As sample sizes grow, researchers are presented with new opportunities to detect and account for grouping factors and covariance structure present in large experimental designs. In particular, standard linear model methods cannot account for the covariance and grouping structures present in large datasets, and the existing linear mixed models (LMM) tools are neither scalable nor exploit the computational speed-ups afforded by vectorisation of computations over voxels. Further, nearly all existing tools for imaging (fixed or mixed effect) do not account for variability in the patterns of missing data near cortical boundaries and the edge of the brain, and instead omit any voxels with any missing data. Yet in the large-n setting, such a voxel-wise deletion missing data strategy leads to severe shrinkage of the final analysis mask. To counter these issues, we describe the "Big" Linear Mixed Models (BLMM) toolbox, an efficient Python package for large-scale fMRI LMM analyses. BLMM is designed for use on high performance computing clusters and utilizes a Fisher Scoring procedure made possible by derivations for the LMM Fisher information matrix and score vectors derived in our previous work, Maullin-Sapey and Nichols (2021).


title: Cellular stress in brain organoids is limited to a distinct and bioinformatically removable subpopulation.

abstract: Organoids enable disease modeling in complex and structured human tissue, in vitro. Like most 3D models, they lack sufficient oxygen supply, leading to cellular stress. These negative effects are particularly prominent in complex models, like brain organoids, where they can prevent proper lineage commitment. Here, we analyze brain organoid and fetal single cell RNA sequencing (scRNAseq) data from published and new datasets totaling over 190,000 cells. We describe a unique stress signature found in all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined organoid cell population, and present Gruffi, an algorithm that uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. Our data show that adverse effects of cell stress can be corrected by bioinformatic analysis, improving developmental trajectories and resemblance to fetal data.


title: A Trem2*R47H mouse model without cryptic splicing drives age- and disease-dependent tissue damage and synaptic loss in response to plaques

abstract: Genome-Wide Association Studies revealed the TREM2 R47H variant as one of the strongest genetic risk factors for late-onset Alzheimers Disease (AD). Unfortunately, many current TREM2*R47H mouse models are associated with cryptic mRNA splicing of the mutant allele that produces a confounding reduction in protein product. We have developed the Trem2R47H NSS (Normal Splice Site) mouse model where the Trem2 allele is expressed at a similar level to the wild-type Trem2 allele, without evidence of cryptic splicing products, and appropriate inflammatory responses to cuprizone challenge. Utilizing the 5xFAD mouse model, we report age- and disease-dependent changes in response to pathology. At an early disease stage (4 mo), homozygous Trem2R47H NSS; hemizygous 5xFAD (Trem2R47H NSS ; 5xFAD) mice have reduced size and number of microglia plus impaired interaction with plaques, that is associated with increased dystrophic neurites and axonal damage detected through plasma neurofilament light chain (NfL) level and suppressed inflammation. However, homozygosity for Trem2R47H NSS suppressed LTP deficits and presynaptic puncta loss caused by the 5xFAD transgene array. At a more advanced disease stage (12 mo,) Trem2R47H NSS ; 5xFAD mice no longer display impaired plaque-microglia interaction or suppressed inflammatory gene expression, although NfL levels remain elevated, and a unique interferon-related gene expression signature is seen. Furthermore, Trem2R47H NSS ; 5xFAD mice also display robust LTP deficits and exacerbated presynaptic loss. Collectively, we provide a Trem2R47H variant mouse without cryptic splicing, and demonstrate it has disease stage dependent effects when combined with a plaque bearing model, with an initial loss of function that ultimately resolves, giving rise to a unique interferon signature and associated tissue damage.