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ALG-2 And Peflin Stimulate or Inhibit COPII Targeting And Secretion In Response To Calcium Signaling ER-to-Golgi transport is the first step in the constitutive secretory pathway which, unlike regulated secretion, is believed to proceed non-stop regardless of Ca2+ flux. Rowever, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adjusting the ER export rate of COPII-sorted cargos up or down by ~50%. At steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. During Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication, following secretory increases, or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Within the NRK cell model, distinct Ca2+ signaling patterns can produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased COPTT outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPTT outer shell and decreased peflin at ERES. PEF protein complexes represent a true regulator of transport as they are dispensable for secretion yet adjust the secretion rate to physiological conditions. Their dynamics affects secretion of important physiological cargoes such as collagen T and significantly impacts ER stress.
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cell biology
|
Plant SYP12 syntaxins mediate an evolutionarily conserved general immunity to filamentous pathogens Filamentous fungal and oomycete plant pathogens that invade by direct penetration through the leaf epidermal cell wall, cause devastating plant diseases. Towards non-adapted filamentous pathogens, plant pre-invasive immunity is highly effective and durable. Pre- and post-invasive immunity correlates with the formation of evolutionarily conserved and cell-autonomous cell wall structures, named papillae and encasements. Yet, it is still unresolved how papillae/encasements are formed and whether these defense structures prevent pathogen ingress. Here we show that in Arabidopsis, the two closely related members of the SYP12 clade of syntaxins (PEN1 and SYP122) are indispensable for the formation of papillae and encasements. Moreover, loss-of-function mutants were hampered in pre-invasive immunity towards a range of taxonomically distinct non-adapted filamentous pathogens, underlining the versatility and efficacy of this defense. Complementation studies using SYP12s from the early diverging land plant, Marchantia polymorpha, showed that the SYP12 clade immunity function has survived 470 My of independent evolution. These results suggest that ancestral land plants evolved the SYP12 clade to provide a broad and durable pre-invasive immunity to facilitate their life on land, and pave the way to a better understanding of how adapted pathogens overcome this ubiquitous plant defense strategy.
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plant biology
|
Predicting and Visualizing STK11 Mutation in Lung Adenocarcinoma Histopathology Slides Using Deep Learning Studies have shown that STK11 mutation plays a critical role in affecting the lung adenocarcinoma (LUAD) tumor immune environment. By training an Inception-Resnet-v2 deep convolutional neural network model, we were able to classify STK11-mutated and wild type LUAD tumor histopathology images with a promising accuracy (per slide AUROC=0.795). Dimensional reduction of the activation maps before the output layer of the test set images revealed that fewer immune cells were accumulated around cancer cells in STK11-mutation cases. Our study demonstrated that deep convolutional network model can automatically identify STK11 mutations based on histopathology slides and confirmed that the immune cell density was the main feature used by the model to distinguish STK11-mutated cases.
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bioinformatics
|
Molecular epidemiology of third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia SynopsisThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using rapid ([~]40 min) real-time PCR assays targeting the most common extended spectrum {beta}-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). AmpC CMY {beta}-lactamase prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one {beta}-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. A subset of isolates (n=98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants, and to verify genotyping accuracy. WGS of {beta}-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.
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microbiology
|
Next-generation phylogeography resolves post-glacial colonization patterns in a widespread carnivore, the red fox (Vulpes vulpes), in Europe. Carnivores tend to exhibit a lack of (or less pronounced) genetic structure at continental scales in both a geographic and temporal sense and this can confound the identification of post-glacial colonization patterns in this group. In this study we used genome-wide data (using Genotyping-by-Sequencing (GBS)) to reconstruct the phylogeographic history of a widespread carnivore, the red fox (Vulpes vulpes), by investigating broad-scale patterns of genomic variation, differentiation and admixture amongst contemporary populations in Europe. Using 15,003 single nucleotide polymorphisms (SNPs) from 524 individuals allowed us to identify the importance of refugial regions for the red fox in terms of endemism (e.g. Iberia). In addition, we tested multiple post-glacial re-colonization scenarios of previously glaciated regions during the Last Glacial Maximum using an Approximate Bayesian Computation (ABC) approach that were unresolved from previous studies. This allowed us to identify the role of admixture from multiple source population post-Younger Dryas in the case of Scandinavia and ancient land-bridges in the colonization of the British Isles. A natural colonization of Ireland was deemed more likely than an ancient human-mediated introduction as has previously been proposed and potentially points to an increased mammalian fauna on the island in the early post-glacial period. Using genome-wide data has allowed us to tease apart broad-scale patterns of structure and diversity in a widespread carnivore in Europe that was not evident from using more limited marker sets and provides a foundation for next-generation phylogeographic studies in other non-model species.
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evolutionary biology
|
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex Cells dynamically adapt organelle size to current physiological demand. Organelle growth requires membrane biogenesis and therefore needs to be coordinated with lipid metabolism. The endoplasmic reticulum (ER) can undergo massive expansion, but the regulatory mechanisms that govern ER membrane biogenesis are largely unclear. Here, we conduct a genetic screen for factors involved in ER membrane expansion in budding yeast and identify the ER transmembrane protein Ice2 as a strong hit. We show that Ice2 promotes ER membrane biogenesis by opposing the phosphatidic acid phosphatase Pah1, which is called lipin in metazoa. Ice2 inhibits the conserved Nem1-Spo7 complex, which dephosphorylates and thus activates Pah1. Furthermore, Ice2 cooperates with the transcriptional regulation of lipid synthesis genes and helps to maintain ER homeostasis during ER stress. These findings establish inhibition of the lipin phosphatase complex as an important mechanism for controlling ER membrane biogenesis.
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cell biology
|
Dynamics of visual perceptual decision-making in freely behaving mice Studying the temporal dynamics of perceptual decisions offers key insights into the cognitive processes contributing to it. Conducting such investigation in a genetically tractable animal model can facilitate the subsequent unpacking of the mechanistic basis of different stages in perceptual dynamics. Here, we investigated the time course as well as fundamental psychophysical constants governing visual perceptual decision-making in freely behaving mice. We did so by analyzing response accuracy against reaction time (i.e., conditional accuracy), in a series of 2-AFC orientation discrimination tasks in which we varied target size, luminance, duration, and presence of a foil. Our results quantified two distinct stages in the time course of mouse visual decision-making - a sensory encoding stage, in which conditional accuracy exhibits a classic tradeoff with response speed, and a subsequent short term memory-dependent stage in which conditional accuracy exhibits a classic asymptotic decay following stimulus offset. We estimated the duration of visual sensory encoding as 200-320 ms across tasks, the lower bound of the duration of short-term memory as ~1700 ms, and the briefest duration of visual stimulus input that is informative as [≤]50 ms. Separately, by varying stimulus onset delay, we demonstrated that the conditional accuracy function and RT distribution can be independently modulated, and found that the duration for which mice naturally withhold from responding is a quantitative metric of impulsivity. Taken together, our results establish a quantitative foundation for investigating the neural circuit bases of visual decision dynamics in mice.
SIGNIFICANCE STATEMENTThis study presents a quantitative breakdown of the time course of visual decision-making in mice during naturalistic behavior. It demonstrates parallel stages in mouse visual perceptual decision dynamics to those in humans, estimates their durations, and shows that mice are able to discriminate well under challenging visual conditions - with stimuli that are brief, low luminance, and small. These results set the stage for investigating the neural bases of visual perceptual decision dynamics and their dysfunction in mice.
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neuroscience
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Early reduction in PD-L1 expression predicts faster treatment response in human cutaneous leishmaniasis Cutaneous leishmaniasis (CL) is a chronic skin disease caused by Leishmania parasites and in Sri Lanka, CL is caused by L. donovani. Pentavalent antimonials (e.g. sodium stibogluconate; SSG) are first line drugs for CL, despite protracted and painful treatment regimens. Data from animal models indicate that the effectiveness of SSG requires drug-immune synergy, but mechanistic insight from patients is lacking. We studied whole blood and lesion transcriptomes from CL patients in Sri Lanka at presentation and during SSG treatment. In lesions, we identified differential expression of immune-related genes, including immune checkpoint molecules, after the onset of treatment whereas no differentially expressed genes were identified in whole blood. We confirmed reduced lesional PD-L1 and IDO1 protein expression on treatment in a second validation cohort, using digital spatial profiling and quantitative immunohistochemistry. Dual IHC-FISH revealed significantly higher expression of these immune checkpoint molecules on parasite-infected compared to non-infected lesional CD68+ monocytes / macrophages. Crucially, early reduction in PD-L1 but not IDO1 expression was predictive of rate of clinical cure and occurred in parallel with a reduction in parasite load. A multivariate cox proportional hazard model showed that patients with lower PD-L1 expression on treatment were more likely to cure earlier (HR= 4.88). Our data support a model whereby the initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition of T cell immunity, facilitating immune-drug synergism and clinical cure. Our findings demonstrate that PD-L1 expression can be used as an early predictor of clinical response to SSG treatment and support the use of PD-L1 inhibition as adjunct host directed therapy in Sri Lankan CL.
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immunology
|
Duchenne Muscular Dystrophy Cell Culture Models Created By CRISPR/Cas9 Gene Editing And Their Application To Drug Screening Gene edition methods are an attractive putative therapeutic option for Duchenne muscular dystrophy and they have an immediate application in the generation of research models. To generate two new edited myoblast cultures that could be useful in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene: DMD{Delta}52-Model, modelling a common Duchenne muscular dystrophy mutation; and in patients immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures and, to show their use in the assessment of DMD treatments, we have performed exon skipping in the DMD{Delta}52-Model and have used the unedited cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMD{Delta}52-Model is limited to the validation to our gene edition protocol, DMD-UTRN-Model offers a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.
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neuroscience
|
Does Vergence Affect Perceived Size? Since Kepler (1604) and Descartes (1637), its been suggested that vergence (the angular rotation of the eyes) plays a key role in size constancy. However, this has never been tested divorced from confounding cues such as changes in the retinal image. In our experiment participants viewed a target which grew or shrank over 5 seconds. At the same time the fixation distance specified by vergence was reduced from 50cm to 25cm. The question was whether the reduction in the viewing distance specified by vergence biased the participants judgements of whether the target grew or shrank? We found no evidence of any bias, and therefore no evidence that eye movements affect perceived size. If this is correct, then this finding has three implications: First, perceived size is much more reliant on cognitive influences than previously thought. This is consistent with the argument that visual scale is purely cognitive in nature (Linton, 2017; 2018). Second, it leads us to question whether the vergence modulation of V1 contributes to size constancy. Third, given the interaction between vergence, proprioception, and the retinal image in the Taylor illusion, it leads us to ask whether this cognitive approach could also be applied to multisensory integration.
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neuroscience
|
Bilateral alignment of receptive fields in the olfactory cortex Each olfactory cortical hemisphere receives ipsilateral odor information directly from the olfactory bulb and contralateral information indirectly from the other cortical hemisphere. Since neural projections to the olfactory cortex are disordered and non-topographic, spatial information cannot be used to align projections from the two sides like in the visual cortex. Therefore, how bilateral information is integrated in individual cortical neurons is unknown. We have found, in mice, that the odor responses of individual neurons to selective stimulation of each of the two nostrils are highly matched, such that odor identity decoding optimized with information arriving from one nostril transfers very well to the other side. Remarkably, these aligned responses are nevertheless asymmetric enough to allow decoding of stimulus laterality. Computational analysis shows that such matched odor tuning is incompatible with purely random connections but is explained readily by Hebbian plasticity structuring bilateral connectivity. Our data reveal that despite the distributed and fragmented sensory representation in the olfactory cortex, odor information across the two hemispheres is highly coordinated.
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neuroscience
|
Transparent and flexible ECoG electrode arrays based on silver nanowire networks for neural recordings This work explored hybrid films of silver nanowires (AgNWs) with indium-zinc oxide (IZO) for developing high-performance and low-cost electrocorticography (ECoG) electrodes. The transparent hybrid films achieved a sheet resistance of 6 {Omega}/sq enabling electrodes with 500 m diameter to reach an impedance of 20 k{Omega} at 1 kHz and a charge storage capacity of 3.2 mC/cm2, an improvement in properties over IZO electrodes, whose performance is on par with the classical tin doped indium oxide (ITO). Characterization of light-induced artifacts was performed showing that light intensities <14 mW/mm2 elicit minimal electrical potential variation, which falls within the magnitude of baseline noise. The validation of electrodes in vivo was achieved by recording electrical neural activity from the surface of rat cortex under anaesthesia. Moreover, the presence of the hybrid films did not cause distortion of light during fluorescence microscopy. This study highlighted the capabilities of the hybrid structure of AgNWs with IZO, that can be fabricated with industrially-established processes at low cost, to be used for transparent ECoG electrodes, offering a new way to record neural electrical activity on a large and fast scale with direct visualization of neurons.
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neuroscience
|
Normalizing and denoising protein expression data from droplet-based single cell profiling Multimodal single-cell protein and transcriptomic profiling (e.g. CITE-seq) holds promise for comprehensive dissection of cellular heterogeneity, yet protein counts measured by oligo-conjugated-antibody can have substantial noise that masks biological variations. Here we integrated experiments and computational analysis to reveal two major noise sources: protein-specific noise from unbound antibodies and cell-specific noise captured by the shared variance of isotype controls and background protein counts. We provide an open source R package (dsb) to denoise and normalize CITE-seq data based on these findings. (https://cran.r-project.org/web/packages/dsb/index.html).
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bioinformatics
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Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in Sugar Porter family The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here we present a 2.4 [A] crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic "Sugar Porter motif" and a conserved "A motif" stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl- ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the Sugar Porter family.
Summary BlurbNew structure of GLUT1 compared to GLUT3 explain different substrate affinities. The result provide a functional rationale for key structural motifs that define the universal Sugar Porter family.
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biochemistry
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Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (-150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying sizes, we assessed the diameter of the water pores in living cells and found that they readily accommodated the passage of 2 nm-wide molecules, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPPs cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.
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cell biology
|
The evolution of mechanisms to divide labour in microorganisms In bacteria and other microorganisms, the cells within a population often show extreme phenotypic variation. Different species use different mechanisms to determine how distinct phenotypes are allocated between individuals, including coordinated, random, and genetic determination. However, it is not clear if this diversity in mechanisms is adaptive--arising because different mechanisms are favoured in different environments--or is merely the result of non-adaptive artifacts of evolution. We use theoretical models to analyse the relative advantages of the two dominant mechanisms to divide labour between reproductives and helpers in microorganisms. We show that coordinated specialisation is more likely to evolve over random specialisation in well-mixed groups when: (i) social groups are small; (ii) helping is more "essential"; and (iii) there is a low metabolic cost to coordination. We find analogous results when we allow for spatial structure with a more detailed model of cellular filaments. More generally, this work shows how diversity in the mechanisms to produce phenotypic heterogeneity could have arisen as adaptations to different environments.
|
evolutionary biology
|
Rapid specialization of counter defenses enables two-spotted spider mite to adapt to novel plant hosts Genetic adaptation, occurring over a long evolutionary time, enables host-specialized herbivores to develop novel resistance traits and to efficiently counteract the defenses of a narrow range of host plants. In contrast, physiological acclimation, leading to the suppression and/or detoxification of host defenses is hypothesized to enable broad-generalists to shift between plant hosts. However, the host adaptation mechanisms used by generalists composed of host-adapted populations are not known. Tetranychus urticae is an extreme generalist herbivore whose individual populations perform well only on a subset of potential hosts. We combined experimental evolution, Arabidopsis genetics, mite reverse genetics, and pharmacological approaches to examine mite host adaptation upon the shift of a bean-adapted population to Arabidopsis thaliana. We showed that cytochrome P450 monooxygenases are required for mite adaptation to Arabidopsis. We identified activities of two tiers of P450s: general xenobiotic-responsive P450s that have a limited contribution to mite adaptation to Arabidopsis and adaptation-associated P450s that efficiently counteract Arabidopsis defenses. In {approx}25 generations of mite selection on Arabidopsis plants, mites evolved highly efficient detoxification-based adaptation, characteristic of specialist herbivores. This demonstrates that specialization to plant resistance traits can occur within the ecological timescale, enabling the two-spotted spider mite to shift to novel plant hosts.
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evolutionary biology
|
Predicting missing links in global host-parasite networks O_LIParasites that infect multiple species cause major health burdens globally, but for many, the full suite of susceptible hosts is unknown. Predicting undocumented host-parasite associations will help expand knowledge of parasite host specificities, promote the development of theory in disease ecology and evolution, and support surveillance of multi-host infectious diseases. Analysis of global species interaction networks allows for leveraging of information across taxa, but link prediction at this scale is often limited by extreme network sparsity, and lack of comparable trait data across species.
C_LIO_LIHere we use recently developed methods to predict missing links in global mammal-parasite networks using readily available data: network properties and evolutionary relationships among hosts. We demonstrate how these link predictions can efficiently guide the collection of species interaction data and increase the completeness of global species interaction networks.
C_LIO_LIWe amalgamate a global mammal host-parasite interaction network (>29,000 interactions) and apply a hierarchical Bayesian approach for link prediction that leverages information on network structure and scaled phylogenetic distances among hosts. We use these predictions to guide targeted literature searches of the most likely yet undocumented interactions, and identify empirical evidence supporting many of the top "missing" links.
C_LIO_LIWe find that link prediction in global host-parasite networks can accurately predict parasites of humans, domesticated animals, and endangered wildlife, representing a combination of published interactions missing from existing global databases, and potential but currently undocumented associations.
C_LIO_LIOur study provides further insight into the use of phylogenies for predicting host-parasite interactions, and highlights the utility of iterated prediction and targeted search to efficiently guide the collection of host-parasite interaction. These data are critical for understanding the evolution of host specificity, and may be used to support disease surveillance through a process of predicting missing links, and targeting research towards the most likely undocumented interactions.
C_LI
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ecology
|
Predicting Endometrial Cancer Subtypes and Molecular Features from Histopathology Images Using Multi-resolution Deep Learning Models The determination of endometrial carcinoma histological subtypes, molecular subtypes, and mutation status is a critical diagnostic process that directly affects patients prognosis and treatment options. Compared to the histopathological approach, however, the availability of molecular subtyping is limited as it can only be accurately obtained by genomic sequencing, which may be cost prohibitive. Here, we implemented a customized multi-resolution deep convolutional neural network, Panoptes, that predicts not only the histological subtypes, but also molecular subtypes and 18 common gene mutations based on digitized H&E stained pathological images. The model achieved high accuracy and generalized well on independent datasets. Our results suggest that Panoptes has potential clinical application of helping pathologists determine molecular subtypes and mutations of endometrial carcinoma without sequencing.
SignificanceRecently, molecular subtyping and mutation status are increasingly utilized in clinical practice as they offer better-informed prognosis and the possibility of individualized therapies for endometrial carcinoma patients. Taking advantage of the multi-resolution nature of the whole slide digital histopathology images, our Panoptes models integrate features of different magnification and make accurate predictions of histological subtypes, molecular subtypes, and key mutations in much faster workflows compared to conventional sequencing-based analyses. Feature extraction and visualization revealed that the model relied on human-interpretable patterns. Overall, our multi-resolution deep learning model is capable of assisting pathologists determine molecular subtypes of endometrial carcinoma, which can potentially accelerate diagnosis process.
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bioinformatics
|
An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. NKX2-1 is required for optimal tumor initiation, but dispensable for growth of established tumors, in BRAFV600E driven disease. NKX2-1-deficient, BRAFV600E driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors also induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.
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cancer biology
|
Expressions for Bayesian confidence of drift diffusion observers in dynamic stimuli tasks Much work has explored the possibility that the drift diffusion model, a model of response times and choices, could be extended to account for confidence reports. Many methods for making predictions from such models exist, although these methods either assume that stimuli are static over the course of a trial, or are computationally expensive, making it difficult to capitalise on trial-by-trial variability in dynamic stimuli. Using the framework of the drift diffusion model with time-dependent thresholds, and the idea of a Bayesian confidence readout, we derive expressions for the probability distribution over confidence reports. In line with current models of confidence, the derivations allow for the accumulation of "pipeline" evidence which has been received but not processed by the time of response, the effect of drift rate variability, and metacognitive noise. The expressions are valid for stimuli which change over the course of a trial with normally distributed fluctuations in the evidence they provide. A number of approximations are made to arrive at the final expressions, and we test all approximations via simulation. The derived expressions only contain a small number of standard functions, and only require evaluating once per trial, making trial-by-trial modelling of confidence data in dynamic stimuli tasks more feasible. We conclude by using the expressions to gain insight into the confidence of optimal observers, and empirically observed patterns.
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animal behavior and cognition
|
KDM3A regulates alternative splicing of cell-cycle genes following DNA damage Changes in the cellular environment result in chromatin structure alteration, which in turn regulates gene expression. To learn about the effect of the cellular environment on the transcriptome, we studied the H3K9 de-methylase KDM3A. Using RNA-seq, we found that KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle and DNA damage. We showed that KDM3A undergoes phosphorylation by PKA at serine 265 following DNA damage, and that the phosphorylation is important for a proper cell cycle regulation. We demonstrated that SAT1 alternative splicing, regulated by KDM3A, plays a role in cell cycle regulation. Furthermore we found that KDM3As demethylase activity is not needed for SAT1 alternative splicing regulation. In addition, we identified KDM3As protein partner ARID1A, the SWI/SNF subunit, and SRSF3 as regulators of SAT1 alternative splicing and showed that KDM3A is essential for SRSF3 binding to SAT1 pre-mRNA. These results suggest that KDM3A serves as a sensor of the environment and an adaptor for splicing factor binding. Our work reveals chromatin sensing of the environment in the regulation of alternative splicing.
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molecular biology
|
The cue-reactivity paradigm: An ensemble of networks driving attention and cognition when viewing drug and natural reward-related stimuli BackgroundThe cue-reactivity paradigm is a widely adopted neuroimaging probe engendering brain activity linked with attentional, affective, and reward processes following presentation of appetitive stimuli. Given the multiple mental operations invoked, we sought to decompose cue-related brain activity into constituent components employing emergent meta-analytic techniques when considering drug and natural reward-related cues.
MethodsWe conducted multiple coordinate-based meta-analyses delineating common and distinct brain activity convergence across cue-reactivity studies (N=196 articles) involving drug (n=133) or natural reward-related (n=63) visual stimuli. Subsequently, we characterized the connectivity profiles of identified brain regions by using them as seeds in task-independent and task-dependent functional connectivity analyses. Using hierarchical clustering on these connectivity profiles, we grouped cue-related brain regions into subnetworks. Functional decoding was then employed to characterize mental operations linked with each subnetwork.
ResultsAcross all studies, pooled activity convergence was observed in the striatum, amygdala, thalamus, cingulate, insula, and multiple frontal, parietal, and occipital regions. Drug-distinct convergence (drug>natural) was observed notably in the posterior cingulate cortex (PCC), dorsolateral prefrontal cortex (dlPFC), and temporal and parietal regions, whereas distinct natural reward convergence (natural>drug) was observed in thalamic, insular, orbitofrontal, and occipital regions. Hierarchical clustering using each regions connectivity profiles identified six subnetworks, involving: 1) occipital and thalamic (lateral geniculate nucleus) regions functionally linked with early visual processing, 2) occipital-temporal regions associated with higher level visual association, 3) parietal-frontal regions linked with cognitive control mechanisms, 4) posterior and ventral insula as well as anterior cingulate cortex (ACC) functionally linked with salient event detection, 5) nucleus accumbens, PCC, precuneus, ACC, and thalamus (mediodorsal) associated with subjective valuation, and 6) bilateral amygdalae, orbitofrontal, and dorsal insula regions linked with affective processes.
ConclusionsThese outcomes suggest multifaceted brain activity during the cue-reactivity paradigm can be decomposed into more elemental processes and indicate that while drugs of abuse usurp the brains natural reward processing system, some regions appear distinctly related to drug cue-reactivity (e.g., PCC, dlPFC).
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neuroscience
|
Autotaxin impedes anti-tumor immunity by suppressing chemotaxis and tumor infiltration of CD8+ T cells Autotaxin (ATX) is secreted by diverse cell types to produce lysophosphatidic acid (LPA) that regulates multiple biological functions via G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis mainly via LPAR1; however, its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T-cell repulsion predominantly involves G12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T-cell responses but suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from patient samples are consistent with intra-tumor ATX acting as a T-cell repellent. These studies highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T-cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.
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cancer biology
|
Quality control and processing of nascent RNA profiling data Nascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable work-flow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using cluster, providing a portable first step for genomic nascent RNA analysis.
AvailabilityBSD2-licensed code and documentation: https://peppro.databio.org.
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bioinformatics
|
Genome editing human primary T cells with microfluidic vortex shedding & CRISPR Cas9 Microfluidic vortex shedding (VS) can rapidly deliver mRNA to T cells with high yield. The mechanistic underpinning of VS intracellular delivery remains undefined and VS-Cas9 genome editing requires further studies. Herein, we evaluated a series of VS devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a VS design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of CRISPR-Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field (eVS). VS alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with eVS with negligible impact on cell viability. Cumulatively, these results demonstrate the utility of VS and eVS for genome editing human primary T cells with Cas9 RNPs.
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immunology
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Improving the validity of neuroimaging decoding tests of invariant and configural neural representation Many research questions in sensory neuroscience involve determining whether the neural representation of a stimulus property is invariant or specific to a particular stimulus context (e.g., Is object representation invariant to translation? Is the representation of a face feature specific to the context of other face features?). Between these two extremes, representations may also be context-tolerant or context-sensitive. Most neuroimaging studies have used operational tests in which a target property is inferred from a significant test against the null hypothesis of the opposite property. For example, the popular cross-classification test concludes that representations are invariant or tolerant when the null hypothesis of specificity is rejected. A recently developed neurocomputational theory provides two insights regarding such tests. First, tests against the null of context-specificity, and for the alternative of context-invariance, are prone to false positives due to the way in which the underlying neural representations are transformed into indirect measurements in neuroimaging studies. Second, jointly performing tests against the nulls of invariance and specificity allows one to reach more precise and valid conclusions about the underlying representations. Here, we provide empirical and computational evidence supporting both of these theoretical insights. In our empirical study, we use encoding of orientation and spatial position in primary visual cortex as a case study, as previous research has established that these properties are encoded in a context-sensitive way. Using fMRI decoding, we show that the cross-classification test produces false-positive conclusions of invariance, but that more valid conclusions can be reached by jointly performing tests against the null of invariance. The results of two simulations further support both of these conclusions. We conclude that more valid inferences about invariance or specificity of neural representations can be reached by jointly testing against both hypotheses, and using neurocomputational theory to guide the interpretation of results.
Author SummaryMany research questions in sensory neuroscience involve determining whether the representation of a stimulus property is invariant or specific to a change in stimulus context (e.g., translation-invariant object representation; configural representation of face features). Between these two extremes, representations may also be context-tolerant or context-sensitive. Most neuroimaging research has studied invariance using operational tests, among which the most widely used in recent years is cross-classification. We provide evidence from a functional MRI study, simulations, and theoretical results supporting two insights regarding such tests: (1) tests that seek to provide evidence for invariance (like cross-classification) have an inflated false positive rate, but (2) using complementary tests that seek evidence for context-specificity leads to more valid conclusions.
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neuroscience
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JACKIE: Enumeration of genomic single- and multi-copy target sites for CRISPR and other engineered nuclease systems ZFP-, TALE-, and CRISPR-based methods for genome, epigenome editing and imaging have provided powerful tools to interrogate functions of genomes. Targeting sequence design is vital to the success of these experiments. While existing design software mainly focus on designing target sequence for specific elements, we report here the implementation of JACKIE (Jackie and Alberts Comprehensive K-mer Instances Enumerator), a suite of software for enumerating all single- and multi-copy sites in the genome that can be incorporated for genome-scale designs as well as loaded onto genome browsers alongside other tracks for convenient web-based graphic-user-interface (GUI)-enabled design. We also implement fast algorithms to identify sequence neighborhoods or off-target counts of targeting sequences so that designs with low probability of off-target can be identified among millions of design sequences in reasonable time. We demonstrate the application of JACKIE-designed CRISPR site clusters for genome imaging.
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bioinformatics
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Clustered cortical inhibition confers robust balance and explains variability dynamics and reaction times during motor task performance Behaving animals have to process partial or incomplete information to plan movements. Neural activity and behavior of highly trained animals are strikingly variable across behavioral trials. In the present study, we show that incomplete information is reflected in both neural and behavioral variability. We study a mechanism for such variability in spiking neural network models with cluster topologies that enables multistability and attractor dynamics. Multistable attractors have been studied in spiking neural networks through clusters of strongly interconnected excitatory neurons. However, we show that existing models suffer from weak robustness and fail to reproduce the experimental findings. To overcome these problems, we introduce a novel architecture with excitatory and inhibitory clusters of spiking neurons. In contrast to earlier prominent models of excitatory clusters, this model ensures the local balance of excitation and inhibition and shows robust multistability across a wide range of network parameters. Furthermore, with the appropriate stimulation of network clusters, this network topology enables qualitatively and quantitatively reproducing in vivo firing rates, variability dynamics and behavioral reaction times for different task conditions as observed in recordings from the motor cortex of behaving monkeys.
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neuroscience
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A meta-analysis of epigenome-wide association studies in Alzheimer's disease highlights novel differentially methylated loci across cortex Epigenome-wide association studies of Alzheimers disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimers disease (N=1,453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N=1,408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further > 600 unique donors. The meta-analysis summary statistics are available in our online data resource (www.epigenomicslab.com/ad-meta-analysis/).
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genomics
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Cumulus cell acetyl-CoA metabolism from acetate is associated with maternal age but only partially with oocyte maturity Cumulus cell (CC) clumps that associate with oocytes provide the oocytes with growth and signaling factors. Thus, the metabolism of the CCs may influence oocyte function and CC metabolism may be predictive of oocyte competence for in vitro fertilization. CCs are thought to be highly glycolytic but data on other potential carbon substrates are lacking in humans. This was a prospective and blinded cohort study that was designed to examine the substrate utilization of CCs by age and oocyte competence. Individual sets of CC clumps from participants were removed after oocyte retrieval procedure, incubated with stable isotope labeled substrates, and analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for isotopologue enrichment of major metabolic intermediates, including acetyl-CoA. The acyl-chain of acetyl-CoA contains 2 carbons that can be derived from 13C-labeled substrates resulting in a M+2 isotopologue that contains 2 13C atoms. Comparing the fate of three major carbon sources, mean enrichment of M+2 acetyl-CoA (mean, standard deviation) was for glucose (3.6, 7.7), for glutamine (9.4, 6.2), and for acetate (20.7, 13.9). Due to this unexpected high and variable labeling from acetate, we then examined acetyl-CoA mean % enrichment from acetate of in 278 CCs from 21 women [≤]34 (49.06, 12.73) decreased with age compared to 124 CCs from 10 women >34 (43.48, 16.20) (p=0.0004, t test). The CCs associated with the immature prophase I oocytes had significantly lower enrichment in M+2 acetyl CoA compared to the CCs associated with the metaphase I and metaphase II oocytes (difference: -6.02, CI: -1.74,-13.79, p=0.013). Acetate metabolism in individual CC clumps was positively correlated with oocyte maturity and decreased with maternal age. These findings indicate that CC metabolism of non-glucose substrates should be investigated relative to oocyte function and age-related fertility.
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biochemistry
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Chromatin dynamics are constrained by loops and driven by the INO80 nucleosome remodeler The chromosomes - DNA polymers and their binding proteins - are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of these polymers to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse-model polymer simulations. We first investigated the model that the activity of two loop extrusion factors, cohesin and condensin, drives chromatin dynamics. Surprisingly, deactivating cohesin or condensin increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. We also find that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taken together we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.
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cell biology
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Visualizing synaptic plasticity in vivo by large-scale imaging of endogenous AMPA receptors Elucidating how synaptic molecules such as AMPA receptors mediate neuronal communication and tracking their dynamic expression during behavior is crucial to understand cognition and disease, but current technological barriers preclude large-scale exploration of molecular dynamics in vivo. We have developed a suite of innovative methodologies that break through these barriers: a new knockin mouse line with fluorescently tagged endogenous AMPA receptors, two-photon imaging of hundreds of thousands of labeled synapses in behaving mice, and computer-vision-based automatic synapse detection. Using these tools, we can longitudinally track how the strength of synapses changes during behavior. We used this approach to generate an unprecedentedly detailed spatiotemporal map of synaptic plasticity underlying sensory experience. More generally, these tools can be used as an optical probe capable of measuring functional synapse strength across entire brain areas during any behavioral paradigm, describing complex system-wide changes with molecular precision.
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neuroscience
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Effects of emotion and semantic relatedness on recognition memory: Behavioral and electrophysiological evidence Some aspects of our memory are enhanced by emotion, whereas other can be unaffected or even hindered. Previous studies report impaired associative memory of emotional content, an effect termed associative "emotional interference". The current study used EEG and an associative recognition paradigm to investigate the cognitive and neural mechanisms associated with this effect. In two experiments, participants studied negative and neutral stimulus-pairs that were either semantically related or unrelated. In Experiment 1 emotions were relevant to the encoding task (valence judgment) whereas in Experiment 2 emotions were irrelevant (familiarity judgment). In a subsequent associative recognition test, EEG was recorded while participants discriminated between intact, rearranged, and new pairs. An associative emotional interference effect was observed in both experiments, but was attenuated for semantically related pairs in Experiment 1, where valence was relevant to the task. Moreover, a modulation of an early associative memory ERP component (300-550 ms) occurred for negative pairs when valence was task-relevant (Experiment 1), but for semantically related pairs when valence was irrelevant (Experiment 2). A later ERP component (550-800 ms) showed a more general pattern, and was observed in all experimental conditions. These results suggest that both valence and semantic relations can act as an organizing principle that promotes associative binding. Their ability to contribute to successful retrieval depends on specific task demands.
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neuroscience
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TLR4-pathway impairs synaptic number and cerebrovascular functions through astrocyte activation following traumatic brain injury Background and purposeActivation of astrocytes contributes to synaptic remodeling, tissue repair and neuronal survival following traumatic brain injury (TBI). However, the mechanisms by which these cells interact to infiltrated inflammatory cells to rewire neuronal networks and repair brain functions remain poorly understood. Here, we explored how TLR4-induced astrocyte activation modified synapses and cerebrovascular integrity following TBI.
Experimental approachWe used pharmacological and genetic approaches to determine how functional astrocyte alterations induced by activation of TLR4-pathway in inflammatory cells regulate synapses and neurovascular unit after TBI. For that, we used calcium imaging, immunofluorescence, flow cytometry, blood-brain barrier (BBB) integrity assessment and molecular and behavioral tools.
Key resultsShortly after a TBI there is a recruitment of excitable and reactive astrocytes mediated by TLR4-pathway activation with detrimental effects on PSD-95/VGlut1 synaptic puncta, BBB integrity and neurological outcome. Pharmacological blockage of the TLR4-pathway with TAK242 partially reverted many of the observed effects. Synapses and BBB recovery after TAK242 administration were not observed in IP3R2-/- mice, indicating that effects of TLR4-inhibition depend on the subsequent astrocyte activation. In addition, TBI increased the astrocytic-protein thrombospondin-1 necessary to induce a synaptic recovery in a sub-acute phase.
Conclusions and implicationsOur data demonstrate that TLR4-mediated signaling, most probably though microglia and/or infiltrated monocyte-astrocyte communication, plays a crucial role in the TBI pathophysiology and that its inhibition prevents synaptic loss and BBB damage accelerating tissue recovery/repair, which might represent a therapeutic potential in CNS injuries and disorders.
Declaration of transparency and scientific rigourThis Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.
Bullet point summaryWhat is already known: O_LIAstrocytes and microglia participate in the early cerebral and synaptic response after traumatic brain injury.
C_LIO_LITLR4 antagonism exerts neuroprotection in acute brain injuries.
C_LI
What this study adds: O_LIAcute astrocyte activation contributes to synaptic loss and BBB breakdown in the acute phase of TBI, and synaptic remodeling in the sub-acute phase.
C_LIO_LIAstrocyte activation is mediated by microglia/infiltrating-monocytes activation through TLR4 receptors.
C_LI
Clinical significance: O_LIInhibition of astrocyte activation through TLR4 antagonism could be a promising option for TBI treatment.
C_LI
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neuroscience
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Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs. Endoplasmic Reticulum (ER) stress is a hallmark of various diseases, which is dealt with through the activation of an adaptive signaling pathway named the Unfolded Protein Response (UPR). This response is mediated by three ER-resident sensors and the most evolutionary conserved, IRE1 signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1 RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through Regulated IRE1- Dependent Decay (RIDD). The balance between these two activities plays an instrumental role in cells life and death decisions upon ER stress. Until now, the biochemical and biological outputs of IRE1 RNase activity have been well documented, however, the precise mechanisms controlling whether IRE1 signaling is adaptive or pro-death (terminal) remain unclear. This prompted us to further investigate those mechanisms and we hypothesized that XBP1 mRNA splicing and RIDD activity could be co-regulated by the IRE1 RNase regulatory network. We showed that a key nexus in this pathway is the tRNA ligase RtcB which, together with IRE1, is responsible for XBP1 mRNA splicing. We demonstrated that RtcB is tyrosine phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we identified RtcB Y306 as a key residue which, when phosphorylated, perturbs RtcB interaction with IRE1, thereby attenuating XBP1 mRNA splicing and favoring RIDD. Our results demonstrate that the IRE1 RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the nature of the stress determines cell adaptive or death outputs.
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biochemistry
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Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals putative regulatory toggles operating in native and iPS-derived retina. We report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from native and iPSC-derived murine retina at four matched developmental stages spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, pseudo-time and RNA velocity analyses, to show that iPSC-derived 3D retinal aggregates broadly recapitulate the native developmental trajectories. However, we show relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in iPSC-derived retina. We generate bulk ATAC-Seq data for native and iPSC-derived murine retina identifying [~]125,000 peaks. We combine single-cell RNA-Seq with ATAC-Seq information and obtain evidence that approximately half the transcription factors that are dynamically regulated during retinal development may act as repressors rather than activators. We propose that sets of activators and repressors with cell-type specific expression constitute "regulatory toggles" that lock cells in distinct transcriptome states underlying differentiation. We provide evidence supporting our hypothesis from the analysis of publicly available single-cell ATAC-Seq data for adult mouse retina. We identify subtle but noteworthy differences in the operation of such toggles between native and iPSC-derived retina particularly for the Etv1, Etv5, Hes1 and Zbtb7a group of transcription factors.
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developmental biology
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A Numerical Representation and Classification of Codons to Investigate Codon Alternation Patterns during Genetic Mutations on Disease Pathogenesis Alteration of amino acid is possible due to mutation in codons that could have potential reasons to occur disease. Effective mutation analysis can help to predict the fate of the diseased individual which can be validated later by in-vitro experiments. It may also help an individual who is asymptomatic but having a particular genetic change for early detection and diagnosis during any terminal diseases. We try to investigate the codon alteration patterns and its impact during mutation for the genes known to be responsible for a particular disease. For our current study, we consider two neurodegenerative diseases, Parkinsons disease and Glaucoma. We use numerical representation of four nucleotides based on the number of hydrogen bonds in their chemical structures and make classification of 64 codons as well as amino acids into three different classes (Strong, Weak and Transition). The entire analysis has been carried out based on these classifications. Our analysis reveals that the strong class codons are highly mutated followed by weak and transition class. We observe that most of the mutations occur in the first or second positions in the codon rather than the third. While looking into the chemical properties of amino acid, we observe that amino acids belong to the aliphatic group are affected most during missense mutations. Our investigation further emphasises that in most of the cases the change in the determinative degree of codon due to mutation is directly proportional to the physical density property. Further calculation of determinative degree of 20 amino acids and getting determinative degree of any arbitrary set of amino acid sequences from them may help biologists to understand the evolutionary information of amino acid occurrence frequencies of in proteins. In addition, our scheme gives a more microscopic and alternative representation of the existing codon table that helps in deciphering interesting codon alteration patterns during mutations in disease pathogenesis.
|
bioinformatics
|
A common gene drive language eases regulatory process and eco-evolutionary extensions Synthetic gene drive technologies aim to spread transgenic constructs into wild populations even when they impose organismal fitness disadvantages. The extraordinary diversity of plausible drive mechanisms and the range of selective parameters they may encounter makes it very difficult to convey their relative predicted properties, particularly where multiple approaches are combined. The sheer number of published manuscripts in this field, experimental and theoretical, the numerous techniques resulting in an explosion in the gene drive vocabulary hinder the regulators point of view. We address this concern by defining a simplified parameter based language of synthetic drives. Employing the classical population dynamics approach, we show that different drive construct (replacement) mechanisms can be condensed and evaluated on an equal footing even where they incorporate multiple replacement drives approaches. Using a common language, it is then possible to compare various model properties, a task desired by regulators and policymakers. The generalization allows us to extend the study of the invasion dynamics of replacement drives analytically and, in a spatial setting, the resilience of the released drive constructs. The derived framework is available as a standalone tool. Besides comparing available drive constructs, our tool is also useful for educational purpose. Users can also explore the evolutionary dynamics of future hypothetical combination drive scenarios. Thus, our results appraise the properties and robustness of drives and provide an intuitive and objective way for risk assessment, informing policies, and enhancing public engagement with proposed and future gene drive approaches.
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evolutionary biology
|
Dynamics of Gut Microbiome, IgA Response and Plasma Metabolome in Development of Pediatric Celiac Disease Celiac disease (CD) is an autoimmune disorder triggered by gluten consumption. To identify the role of gut microbes in CD onset, we performed a longitudinal study focusing on two important phases of gut microbiota development at ages 2.5 and 5 (n=16). We obtained samples from children who developed CD during or after the study (CD progressors) and age, sex, and HLA-matched healthy controls. CD progressors had a distinct gut microbiota composition and IgA-sequencing identified unique IgA targets in the gut. Three cytokines, one chemokine, and 19 plasma metabolites were significantly altered in CD progressors at age 5. Feeding C57BL/6J mice with taurodeoxycholic acid (TDCA), a 2-fold increased microbiota-derived metabolite in CD progressors, caused villous atrophy, increased intraepithelial lymphocytes (IELs), CD4+ T-cells, Natural Killer cells, and Qa-1 expression on T-cells while decreasing T-regulatory cells in IELs. Thus, TDCA drives inflammation in the small intestines that potentially contribute to the CD onset.
HighlightsO_LICD progressors have a distinct gut microbiome composition compared to healthy controls in two important phases of gut microbiota development (age 2.5 and 5 years)
C_LIO_LICD progressors have more IgA-coated bacteria in their gut at age 5 compared to healthy controls. Further, IgA-sequencing identified unique bacterial targets in CD progressors.
C_LIO_LIThree plasma proinflammatory cytokines and a chemokine were increased in CD progressors years before diagnosis, indicating an early inflammatory response.
C_LIO_LIWe identified 19 metabolites that are significantly altered in CD progress at age 5 and microbiota-derived TDCA increased two-fold.
C_LIO_LITDCA treatment in B6 mice increased CD4+ cells and NK cells while decreasing CD8+ T-regulatory (Treg) cells. It also increased Qa-1 expression on immune cells.
C_LI
|
immunology
|
A rank-normalized archaeal taxonomy based on genome phylogeny resolves widespread incomplete and uneven classifications An increasing wealth of genomic data from cultured and uncultured microorganisms provides the opportunity to develop a systematic taxonomy based on evolutionary relationships. Here we propose a standardized archaeal taxonomy, as part of the Genome Taxonomy Database (GTDB), derived from a 122 concatenated protein phylogeny that resolves polyphyletic groups and normalizes ranks based on relative evolutionary divergence (RED). The resulting archaeal taxonomy is stable under a range of phylogenetic variables, including marker genes, inference methods, corrections for rate heterogeneity and compositional bias, tree rooting scenarios, and expansion of the genome database. Rank normalization was shown to robustly correct for substitution rates varying up to 30-fold using simulated datasets. Taxonomic curation follows the rules of the International Code of Nomenclature of Prokaryotes (ICNP) while taking into account proposals to formally recognise the rank of phylum and to use genome sequences as type material. The taxonomy is based on 2,392 quality screened archaeal genomes, the great majority of which (93.3%) required one or more changes to their existing taxonomy, mostly as a result of incomplete classification. In total, 16 archaeal phyla are described, including reclassification of three major monophyletic units from the former Euryarchaeota and one phylum resulting from uniting the TACK superphylum into a single phylum. The taxonomy is publicly available at the GTDB website (https://gtdb.ecogenomic.org).
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microbiology
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Corticospinal excitability remains unchanged in the presence of residual force enhancement and does not contribute to increased torque production Following active muscle stretch, muscle force is enhanced, which is known as residual force enhancement (rFE). As earlier studies found apparent corticospinal excitability modulations in the presence of rFE, this study aimed to test whether corticospinal excitability modulations contribute to rFE. Fourteen participants performed submaximal plantar flexion stretch-hold and fixed-end contractions at 30% of their maximal voluntary soleus muscle activity in a dynamometer. During the steady state of the contractions, participants either received subthreshold or suprathreshold transcranial magnetic stimulation (TMS) of their motor cortex, while triceps surae muscle responses to stimulation were obtained via electromyography (EMG), and net ankle joint torque was recorded. B-mode ultrasound imaging was used to confirm muscle fascicle stretch during stretch-hold contractions in a subset of participants. Following stretch of the plantar flexors, an average rFE of 7% and 11% was observed for contractions with subthreshold and suprathreshold TMS, respectively. 41-46 ms following subthreshold TMS, triceps surae muscle activity was suppressed by 19-25%, but suppression was not significantly different between stretch-hold and fixed-end contractions. Similarly, the reduction in plantar flexion torque following subthreshold TMS was not significantly different between contraction conditions. Motor evoked potentials, silent periods and superimposed twitches following suprathreshold TMS were also not significantly different between contraction conditions. As TMS of the motor cortex did not result in any differences between stretch-hold and fixed-end contractions, we conclude that rFE is not linked to changes in corticospinal excitability, making rFE a muscle mechanical property rather than a combined neuro-muscular property.
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neuroscience
|
A framework to identify structured behavioral patterns within rodent spatial trajectories Animal behavior is highly structured. Yet, structured behavioral patterns - or "statistical ethograms" - are not immediately apparent from the full spatiotemporal data that behavioral scientists usually collect. Here, we introduce a framework to quantitatively characterize rodent behavior during spatial (e.g., maze) navigation, in terms of movement building blocks or motor primitives. The hypothesis that we pursue is that rodent behavior is characterized by a small number of motor primitives, which are combined over time to produce open-ended movements. We assume motor primitives to be organized in terms of two sparsity principles: each movement is controlled using a limited subset of motor primitives (sparse superposition) and each primitive is active only for time-limited, time-contiguous portions of movements (sparse activity). We formalize this hypothesis using a sparse dictionary learning method, which we use to extract motor primitives from rodent position and velocity data collected during spatial navigation, and successively to reconstruct past trajectories and predict novel ones. Three main results validate our approach. First, rodent behavioral trajectories are robustly reconstructed from incomplete data, performing better than approaches based on standard dimensionality reduction methods, such as principal component analysis, or single sparsity. Second, the motor primitives extracted during one experimental session generalize and afford the accurate reconstruction of rodent behavior across successive experimental sessions in the same or in modified mazes. Third, in our approach the number of motor primitives associated with each maze correlates with independent measures of maze complexity, hence showing that our formalism is sensitive to essential aspects of task structure. The framework introduced here can be used by behavioral scientists and neuroscientists as an aid for behavioral and neural data analysis. Indeed, the extracted motor primitives enable the quantitative characterization of the complexity and similarity between different mazes and behavioral patterns across multiple trials (i.e., habit formation). We provide example uses of this computational framework, showing how it can be used to identify behavioural effects of maze complexity, analyze stereotyped behavior, classify behavioral choices and predict place and grid cell displacement in novel environments.
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animal behavior and cognition
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Ongoing exposure to peritoneal dialysis fluid alters resident peritoneal macrophage phenotype and activation propensity Peritoneal dialysis (PD) is a more continuous alternative to haemodialysis, for patients with chronic kidney disease, with considerable initial benefits for survival, patient independence and healthcare costs. However, longterm PD is associated with significant pathology, negating the positive effects over haemodialysis. Importantly, peritonitis and activation of macrophages is closely associated with disease progression and treatment failure. However, recent advances in macrophage biology suggest opposite functions for macrophages of different cellular origins. While monocyte-derived macrophages promote disease progression in some models of fibrosis, tissue resident macrophages have rather been associated with protective roles. Thus, we aimed to identify the relative contribution of tissue resident macrophages to PD induced inflammation in mice. Unexpectedly, we found an incremental loss of homeostatic characteristics, anti-inflammatory and efferocytic functionality in peritoneal resident macrophages, accompanied by enhanced inflammatory responses to external stimuli. Moreover, presence of glucose degradation products within the dialysis fluid led to markedly enhanced inflammation and almost complete disappearance of tissue resident cells. Thus, alterations in tissue resident macrophages may render longterm PD patients sensitive to developing peritonitis and consequently fibrosis/sclerosis.
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immunology
|
A Histoplasma capsulatum lipid metabolic map identifies antifungal targets and virulence factors Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here we built a comprehensive map of the Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development.
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microbiology
|
Chromatin remodelling by INO80 at transcription pause sites promotes premature termination of mRNA synthesis How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process.
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molecular biology
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Brain gene co-expression networks link complement signaling with convergent synaptic pathology in schizophrenia The most significant common variant association for schizophrenia (SCZ) reflects increased expression of the complement component 4A (C4A). Yet, it remains unclear how C4A interacts with other SCZ risk genes and whether the complement system is more broadly implicated in SCZ pathogenesis. Here, we integrate several existing, large-scale genetic and transcriptomic datasets to interrogate the functional role of the complement system and C4A in the human brain. Surprisingly, we find no significant genetic enrichment among known complement system genes for SCZ. Conversely, brain co-expression network analyses using C4A as a seed gene revealed that genes down-regulated when C4A expression increased exhibit strong and specific genetic enrichment for SCZ risk. This convergent genomic signal reflected neuronal, synaptic processes and was sexually dimorphic and most prominent in frontal cortical brain regions. Overall, these results indicate that synaptic pathways--rather than the complement system--are the driving force conferring SCZ risk.
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genomics
|
Combining tensor decomposition and time warping models for multi-neuronal spike train analysis Recordings from large neural populations are becoming an increasingly popular and accessible method in experimental neuroscience. While the activity of individual neurons is often too stochastic to interrogate circuit function on a moment-by-moment basis, multi-neuronal recordings enable us to do so by pooling statistical power across many cells. For example, groups of neurons often exhibit correlated gain or amplitude modulation across trials, which can be statistically formalized in a tensor decomposition framework (Williams et al. 2018). Additionally, the time course of neural population dynamics can be shifted or stretched/compressed, which can be modeled by time warping methods (Williams et al. 2020). Here, I describe how these two modeling frameworks can be combined, and show some evidence that doing so can be highly advantageous for practical neural data analysis--for example, the presence of random time shifts hampers the performance and interpretability of tensor decomposition, while a time-shifted variant of this model corrects for these disruptions and uncovers ground truth structure in simulated data.
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neuroscience
|
TPX2-dependent spindle positioning dictates division site during asymmetric cell division in moss gametophores Asymmetric cell division (ACD) underlies the development of multicellular organisms. It is considered that the division site in land plants is predetermined prior to mitosis and that the localization of the mitotic spindle does not govern the division plane. This contrasts with animal ACD, in which the division site is defined by active spindle-positioning mechanisms. Here, we isolated a hypomorphic mutant of the conserved microtubule-associated protein TPX2 in the moss Physcomitrium patens and observed abnormal spindle motility during cell division. This defect compromised the position of the division site and produced inverted daughter cell sizes in the first ACD of gametophore (leafy shoot) development. The phenotype was rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we have identified an active spindle-positioning mechanism that, reminiscent of acentrosomal ACD in animals, involves microtubules and actin filaments and sets the division site in plants.
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plant biology
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Low frequency traveling waves in the human cortex coordinate neural activity across spatial scales Traveling waves of oscillatory activity are thought to influence neuronal spiking, providing a spatiotemporal frame-work for neural communication. However, no direct link has been established between traveling waves and neuronal spiking in humans. We examined traveling waves in the human lateral temporal lobe by using recordings from intracranial electrodes implanted in twenty participants for seizure monitoring as they performed a paired-associates verbal memory task. We observed ubiquitous low frequency traveling waves across the temporal lobe. While wave occurrence in a broad low frequency range did not differ between successful and unsuccessful memory conditions, in a subset of participants with microelectrode recordings, we found that macro-scale waves co-occurred with micro-scale waves, which in turn were temporally locked to single unit spiking. This temporal coordination between traveling waves at different spatial scales and between waves and neuronal spiking in the human brain suggests a role for traveling waves in neural communication.
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neuroscience
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Simulating future rewards: exploring the impacts of implicit context association and arithmetic booster in delay discounting People have higher preference for immediate over delayed rewards, and it is suggested that such an impulsive tendency is governed by ones ability to simulate future rewards. Consistent with this view, recent studies have shown that enforcing individuals to focus on episodic future thoughts reduces their impulsivity. Inspired by these successful reports, we hypothesized that administration of a simple cognitive task which is linked with future thinking might also function as an effective tool for modulating individuals preference for immediate (or delayed) rewards. Specifically, we used one associative memory task and one working memory task that each of which was administered to intervene acquired amount of information and individuals ability to construct a coherent future event, respectively. Among the set of cognitive tasks, we found that only the arithmetic working memory task had a significant effect of reducing individuals impulsivity. However, in our follow-up experiment, this result was not replicated. Across these two independent experiments, we observed a significant main effect of repetition in individuals impulsivity measure, such that participants showed more patient choices at the second compared to the first assessment task. In conclusion, there was no clear evidence supporting that our suggested intervention tasks effectively reduce individuals impulsivity, while the current results call attention to the importance of taking into account task repetition effects in studying the impacts of cognitive training and intervention.
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neuroscience
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The gliopeptide ODN, a ligand for the benzodiazepine site of GABAA receptors, boosts functional recovery after stroke Following stroke, the survival of neurons and their ability to re-establish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAAR). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the sub-optimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which make it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that during the sub-acute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.
SIGNIFICANCE STATEMENTStroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide (ODN), can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.
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neuroscience
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The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Its mechanism of cell exit is unusual amongst Rickettsiaceae, as Ot buds off the surface of infected cells enveloped in plasma membrane. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, secreted bacterial effectors, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and macropinocytosis, whereas entry of EB Ot is only sensitive to a macropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen.
Author SummaryOrientia tsutsugamushi (Ot) is a bacterial pathogen that causes scrub typhus, a mite-transmitted human disease. This illness is traditionally known to be endemic in the Asia-Pacific, but recent reports of Orientia-like organisms from the Middle East, Africa, and Latin America suggest that it may be globally distributed. Scrub typhus is associated with high mortality if not treated promptly with appropriate antibiotics. Ot is a highly specialized bacterium that can only replicate within living cells, either within the mite vector or in mammalian or human hosts. Ot exits infected cells using a unique mechanism that involves budding off the surface of infected cells. We have discovered that this unusual aspect of its lifecycle involves the bacteria themselves differentiating into a distinct growth form. Different growth forms have not been described in other members of the family Rickettsiaceae, and no other family members have been shown to bud out of host cells in a manner similar to Ot. We find that the two forms of Ot, which we refer to as intracellular and extracellular bacteria (IB and EB respectively), differ in physical properties and protein expression and infect cells through different mechanisms. The identification of structurally and functionally distinct forms of Ot elucidates a vital aspect of this pathogens intracellular life cycle. The two forms are likely to have different antibiotic susceptibilities, therefore our findings may advance the development of novel interventions aimed at inhibiting Ot growth in scrub typhus patients.
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microbiology
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Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle domain subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.
|
plant biology
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CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.
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genomics
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Effects of arousal and movement on secondary somatosensory and visual thalamus All neocortical sensory areas have an associated primary and secondary thalamic nucleus. While the primary nuclei encode sensory information for transmission to cortex, the nature of information encoded in secondary nuclei is poorly understood. We recorded juxtasomally from neurons in secondary somatosensory (POm) and visual (LP) thalamic nuclei of awake head-fixed mice with simultaneous whisker tracking and pupilometry. POm activity correlated with whether or not a mouse was whisking, but not precise whisking kinematics. This coarse movement modulation persisted after unilateral paralysis of the whisker pad and thus was not due to sensory reafference. POm continued to track whisking even during optogenetic silencing of primary somatosensory and motor cortex and after lesion of superior colliculus, indicating that motor efference copy cannot explain the correlation between movement and POm activity. Whisking and pupil dilation were strongly correlated, raising the possibility that POm may track arousal rather than movement. LP, being part of the visual system, is not expected to encode whisker movement. We discovered, however, that LP and POm track whisking equally well, suggesting a global effect of arousal on both nuclei. We conclude that secondary thalamus is a monitor of behavioral state, rather than movement, and may exist to alter cortical activity accordingly.
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neuroscience
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Restoration of motor-evoked cortical activity is a distinguishing feature of the most effective rehabilitation therapy after stroke BackgroundAn ischemic stroke is followed by the remapping of motor representation and extensive changes in cortical excitability involving both hemispheres. Although stimulation of the ipsilesional motor cortex, especially when paired with motor training, facilitates plasticity and functional restoration, the mechanisms underneath the reshaping of cortical functionality are widely unknown.
ObjectiveWe investigated the spatio-temporal features of motor-evoked cortical activity associated with generalized recovery after stroke, and its dependence on the type of rehabilitative treatment.
MethodsWe designed a novel rehabilitative treatment that combines neuro-plasticizing intervention with motor training. Specifically, optogenetic stimulation of peri-infarct excitatory neurons expressing Channelrhodopsin-2 was associated with daily motor training on a robotic device. The effectiveness of the combined therapy was compared with spontaneous recovery and with the single treatments (i.e. individually administered optogenetic stimulation or motor training).
ResultsWe found that only the combined therapy promotes generalized recovery of forelimb function and the rescue of spatio-temporal features of motor-evoked activity. Generalized recovery results from a new excitatory/inhibitory balance between hemispheres as revealed by the augmented motor response flanked by the increased expression of parvalbumin positive neurons in the peri-infarct area.
ConclusionsOur findings demonstrate that though behavioral recovery is not necessarily associated with the restoration of pre-stroke motor-evoked activity, the reestablishment of pre-stroke activation transients was a distinguishing feature of the most efficient therapeutic approach, the combined therapy.
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neuroscience
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Strong effect of Penicillium roqueforti populations on volatile and metabolic compounds responsible for aromas, flavor and texture in blue cheeses Studies of food microorganism domestication can provide important insight into adaptation mechanisms and lead to commercial applications. Penicillium roqueforti is a fungus with four genetically differentiated populations, two of which were independently domesticated for blue cheese-making, with the other two populations thriving in other environments. Most blue cheeses are made with strains from a single P. roqueforti population, whereas Roquefort cheeses are inoculated with strains from a second population. We made blue cheeses in accordance with the production specifications for Roquefort-type cheeses, inoculating each cheese with a single P. roqueforti strain, using a total of three strains from each of the four populations. We investigated differences between the cheeses made with the strains from the four P. roqueforti populations, in terms of the induced flora, the proportion of blue color, water activity and the identity and abundance of aqueous and organic metabolites as proxies for proteolysis and lipolysis as well as volatile compounds responsible for flavor and aroma. We found that the population-of-origin of the P. roqueforti strains used for inoculation had a minor impact on bacterial diversity and no effect on the abundance of the main microorganism. The cheeses produced with P. roqueforti strains from cheese populations had a higher percentage of blue area and a higher abundance of the volatile compounds typical of blue cheeses, such as methyl ketones and secondary alcohols. In particular, the Roquefort strains produced higher amounts of these aromatic compounds, partly due to more efficient proteolysis and lipolysis. The Roquefort strains also led to cheeses with a lower water availability, an important feature for preventing spoilage in blue cheeses, which is subject to controls for the sale of Roquefort cheese. The typical appearance and flavors of blue cheeses thus result from human selection on P. roqueforti, leading to the acquisition of specific features by the two cheese populations. These findings have important implications for our understanding of adaptation and domestication, and for cheese improvement.
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evolutionary biology
|
Transcriptional, epigenetic and metabolic signatures in cardiometabolic syndrome defined by extreme phenotypes. Improving the understanding of cardiometabolic syndrome pathophysiology and its relationship with thrombosis are ongoing healthcare challenges. Using plasma biomarkers analysis coupled with the transcriptional and epigenetic characterisation of cell types involved in thrombosis, obtained from two extreme phenotype groups (obese and lipodystrophy) and comparing these to lean individuals and blood donors, the present study identifies the molecular mechanisms at play, highlighting patterns of abnormal activation in innate immune phagocytic cells and shows that extreme phenotype groups could be distinguished from lean individuals, and from each other, across all data layers. The characterisation of the same obese group, six months after bariatric surgery shows the loss of the patterns of abnormal activation of innate immune cells previously observed. However, rather than reverting to the gene expression landscape of lean individuals, this occurs via the establishment of novel gene expression landscapes. Netosis and its control mechanisms emerge amongst the pathways that show an improvement after surgical intervention. Taken together, by integrating across data layers, the observed molecular and metabolic differences form a disease signature that is able to discriminate, amongst the blood donors, those individuals with a higher likelihood of having cardiometabolic syndrome, even when not presenting with the classic features.
|
genomics
|
Exploring the evolution and adaptive role of mosaic aneuploidy in a clonal Leishmania donovani population using high throughput single-cell genome sequencing Maintenance of stable ploidy over continuous mitotic events is a paradigm for most higher eukaryotes. Defects in chromosome segregation and/or replication can lead to aneuploidy, a condition often considered deleterious. However, in Leishmania, a Protozoan parasite, aneuploidy is a constitutive feature, where variations of somies represent a mechanism of gene expression adaptation, possibly impacting phenotypes. Strikingly, clonal Leishmania populations display cell-to-cell somy variation, a phenomenon named mosaic aneuploidy (MA). However, until recently, no method was available for the determination of the complete karyotype of single Leishmania parasites. To overcome this limitation, we used here for the first time a high-throughput single-cell genomic sequencing (SCGS) method to estimate individual karyotypes of 1560 promastigote cells in a clonal population of Leishmania donovani. We identified 128 different karyotypes, of which 4 were dominant. A network analysis revealed that most karyotypes are linked to each other by changes in copy number of a single chromosome and allowed us to propose a hypothesis of MA evolution. Moreover, aneuploidy patterns that were previously described by Bulk Genome Sequencing as emerging during first contact of promastigotes populations with different drugs are already pre-existing in single karyotypes in the SCGS data, suggesting a (pre-)adaptive role of MA. Additionally, the degree of somy variation was chromosome-specific. The SCGS also revealed a small fraction of cells where one or more chromosomes were nullisomic. Together, these results demonstrate the power of SCGS to resolve sub-clonal karyotype heterogeneity in Leishmania and pave the way for understanding the role of MA in these parasites adaptability.
Update: 25th May 2021A revision of the present preprint was released in BioRxiv on 11th May 2021 (https://www.biorxiv.org/content/10.1101/2021.05.11.443577v2). In the new version, we included two extra samples in our single-cell genome sequencing (SCGS) analysis - the BPK081 cl8 clone (a nearly euploid strain), and a population consisting of a mixture of four L. donovani strains which was used as control for high levels of mosaicism in aneuploidy and for estimation of doublets. We also upgraded the bioinformatics pipeline to determine single-cell karyotypes and performed new fluorescence in situ hybridization (FISH) analysis. The new findings observed especially in the BPK081 cl8 led to a reformulation of the text, a new hypothesis for the evolution of mosaicism and a general restructuring of the article. Therefore, the present preprint is obsolete. Please refer to the new preprint entitled "High throughput single cell genome sequencing gives insights in the generation and evolution of mosaic aneuploidy in Leishmania donovani" for more information.
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genomics
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Comprehensive in virio structure probing analysis of the influenza A virus identifies a functional RNA structure involved in replication and segment interactions The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures on vRNA are thought to be involved in the viral proliferation process, such as intersegment interactions that are necessary for segment bundling. However, the functional RNA structure on vRNA is not well known because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins in a sequence-independent manner. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-seq. By integrating DMS-seq and SHAPE-seq analyses with robust statistical analysis, we inferred quite a few bases paired regions including a pseudoknot structure on segment 5. Notably, when cells were infected with the recombinant virus which had mutations in the pseudoknot structure, the impairment of replication and packaging was observed on the other specific segment. Moreover, we analyzed the comprehensive intersegment RNA interactions in virion by ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq). Our LIGR-seq analysis revealed that the intersegment interactions of the specific segment became less frequent and rearranged in the recombinant virus in concordance with the strength of genome packaging impairment. Our data provide evidence that the functional RNA structure motif on the influenza A virus genome can affect the efficiency of replication and segment bundling through the segment interactions.
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microbiology
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Bridging the gap: Using reservoir ecology and human serosurveys to estimate Lassa virus spillover in West Africa Forecasting the risk of pathogen spillover from reservoir populations of wild or domestic animals is essential for the effective deployment of interventions such as wildlife vaccination or culling. Due to the sporadic nature of spillover events and limited availability of data, developing and validating robust, spatially explicit, predictions is challenging. Recent efforts have begun to make progress in this direction by capitalizing on machine learning methodologies. An important weakness of existing approaches, however, is that they generally rely on combining human and reservoir infection data during the training process and thus conflate risk attributable to the prevalence of the pathogen in the reservoir population with the risk attributed to the realized rate of spillover into the human population. Because effective planning of interventions requires that these components of risk be disentangled, we developed a multi-layer machine learning framework that separates these processes. Our approach begins by training models to predict the geographic range of the primary reservoir and the subset of this range in which the pathogen occurs. The spillover risk predicted by the product of these reservoir specific models is then fit to data on realized patterns of historical spillover into the human population. The result is a geographically specific spillover risk forecast that can be easily decomposed and used to guide effective intervention. Applying our method to Lassa virus, a zoonotic pathogen that regularly spills over into the human population across West Africa, results in a model that explains a modest but statistically significant portion of geographic variation in historical patterns of spillover. When combined with a mechanistic mathematical model of infection dynamics, our spillover risk model predicts that 897,700 humans are infected by Lassa virus each year across West Africa, with Nigeria accounting for more than half of these human infections.
Author SummaryThe 2019 emergence of SARS-CoV-2 is a grim reminder of the threat animal-borne pathogens pose to human health. Even prior to SARS-CoV-2, the spillover of pathogens from animal reservoirs was a persistent problem, with pathogens such as Ebola, Nipah, and Lassa regularly but unpredictably causing outbreaks. Machine-learning models that anticipate when and where pathogen transmission from animals to humans is likely to occur would help guide surveillance efforts and preemptive countermeasures like information campaigns or vaccination programs. We develop a novel machine learning framework that uses datasets describing the distribution of a virus within its host and the range of its animal host, along with data on spatial patterns of human immunity, to infer rates of animal-to-human transmission across a region. By training the model on data from the animal host alone, our framework allows rigorous validation of spillover predictions using human data. We apply our framework to Lassa fever, a viral disease of West Africa that is spread to humans by rodents, and use the predictions to update estimates of Lassa virus infections in humans. Our results suggest that Nigeria is most at risk for the emergence of Lassa virus, and should be prioritized for outbreak-surveillance.
|
ecology
|
Differential Regulation of Single Microtubules and Bundles by a Three-Protein Module A remarkable feature of the microtubule cytoskeleton is co-existence of sub-populations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1, and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find their collective activity promotes elongation of single microtubules, while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (i) Strong rescue activity of CLASP1, which overcomes weaker effects of Kif4A on single microtubules, (ii) Lower microtubule and PRC1-binding affinity of CLASP1, which permit dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule lengths, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.
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cell biology
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A bivariate zero-inflated negative binomial model and its applications to biomedical settings SO_SCPLOWUMMARYC_SCPLOWThe zero-inflated negative binomial (ZINB) distribution has been widely used for count data analyses in various biomedical settings due to its capacity of modeling excess zeros and overdispersion. When there are correlated count variables, a bivariate model is essential for understanding their full distributional features. For this purpose, we develop a Bivariate Zero-Inflated Negative Binomial (BZINB) model that has a simple latent variable framework and parameters with intuitive interpretations. Using this model, we examine two biomedical data examples where the counts are zero-inflated--single cell RNA sequencing (scRNA-seq) data and dental caries count indices. In scRNA-seq data example, a correlation between a pair of genes is estimated after adjusting for the effects of dropout events represented by excess zeros. In the dental caries data, we analyze how the treatment with Xylitol mints affects the marginal mean and other patterns of response manifested in the two dental caries traits. An R package bzinb is available on CRAN.
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genomics
|
Long-read error correction: a survey and qualitative comparison Third generation sequencing technologies Pacific Biosciences and Oxford Nanopore Technologies were respectively made available in 2011 and 2014. In contrast with second generation sequencing technologies such as Illumina, these new technologies allow the sequencing of long reads of tens to hundreds of kbp. These so called long reads are particularly promising, and are especially expected to solve various problems such as contig and haplotype assembly or scaffolding, for instance. However, these reads are also much more error prone than second generation reads, and display error rates reaching 10 to 30%, according to the sequencing technology and to the version of the chemistry. Moreover, these errors are mainly composed of insertions and deletions, whereas most errors are substitutions in Illumina reads. As a result, long reads require efficient error correction, and a plethora of error correction tools, directly targeted at these reads, were developed in the past ten years. These methods can adopt a hybrid approach, using complementary short reads to perform correction, or a self-correction approach, only making use of the information contained in the long reads sequences. Both these approaches make use of various strategies such as multiple sequence alignment, de Bruijn graphs, Hidden Markov Models, or even combine different strategies. In this paper, we describe a complete survey of long-read error correction, reviewing all the different methodologies and tools existing up to date, for both hybrid and self-correction. Moreover, the long reads characteristics, such as sequencing depth, length, error rate, or even sequencing technology, have huge impacts on how well a given tool or strategy performs, and can thus drastically reduce the correction quality. We thus also present an in-depth benchmark of available long-read error correction tools, on a wide variety of datasets, composed of both simulated and real data, with various error rates, coverages, and read lengths, ranging from small bacterial to large mammal genomes.
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bioinformatics
|
Oligomeric amyloid beta prevents myelination in a clusterin-dependent manner White matter loss has been described as a common occurrence in Alzheimers disease (AD) patients for multiple decades. However, it remains unclear why oligodendrocyte progenitor cells (OPCs) fail to repair myelin deficits in these patients. Here, we show that clusterin, a risk factor for late-onset AD, is produced by OPCs and inhibits their differentiation into oligodendrocytes. Specifically, we demonstrate that a unique subset of OPCs produces clusterin. We show that phagocytosis of debris, including amyloid beta (A{beta}) and myelin, drives the upregulation of clusterin in OPCs. We confirm, in vivo, that A{beta} oligomers drive clusterin upregulation and that OPCs phagocytose A{beta}. Furthermore, we show that clusterin is a potent inhibitor of OPC differentiation and prevents the production of myelin proteins. Finally, we demonstrate that clusterin inhibits OPC differentiation by significantly reducing the production of IL-9 by OPCs. Our data reveals that clusterin may be responsible for the lack of myelin repair observed in AD and is a promising therapeutic target for AD-associated cognitive decline.
|
neuroscience
|
A fast and efficient smoothing approach to Lasso regression and an application in statistical genetics: polygenic risk scores for chronic obstructive pulmonary disease (COPD) High dimensional linear regression problems are often fitted using Lasso approaches. Although the Lasso objective function is convex, it is not differentiable everywhere, making the use of gradient descent methods for minimization not straightforward. To avoid this technical issue, we apply Nesterov smoothing to the original (unsmoothed) Lasso objective function. We introduce a closed-form smoothed Lasso which preserves the convexity of the Lasso function, is uniformly close to the unsmoothed Lasso, and allows us to obtain closed-form derivatives everywhere for efficient and fast minimization via gradient descent. Our simulation studies are focused on polygenic risk scores using genetic data from a genome-wide association study (GWAS) for chronic obstructive pulmonary disease (COPD). We compare accuracy and run-time of our approach to the current gold standard in the literature, the FISTA algorithm. Our results suggest that the proposed methodology provides estimates with equal or higher accuracy than the FISTA algorithm while having the same asymptotic runtime scaling. The proposed methodology is implemented in the R-package smoothedLasso, available on the Comprehensive R Archive Network (CRAN).
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bioinformatics
|
Homologous organization of cerebellar pathways to sensory, motor, and associative forebrain Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here we quantify pathways between cerebellum and forebrain using transsynaptic tracing viruses and a whole-brain quantitative analysis pipeline. Retrograde tracing found a majority of descending paths originating from somatomotor cortex. Anterograde tracing of ascending paths encompassed most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei; in neocortex, sensorimotor regions contained the most labeled neurons, but higher densities were found in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlated with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of cerebellar cortex to diverse forebrain targets. We conclude that shared areas of cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function.
Graphical Abstract
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neuroscience
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Intergenerational transmission of the patterns of functional and structural brain networks There is clear evidence of intergenerational transmission of life values, cognitive traits, psychiatric disorders, and even aspects of daily decision making. To investigate biological substrates of this phenomenon, brain has received increasing attention as a measurable biomarker and potential target for intervention. However, no previous study has quantitatively and comprehensively investigated the effects of intergenerational transmission on functional and structural brain networks from parents to their children. Here, by employing an unusually large cohort dataset, we show that patterns of functional and structural brain networks are preserved over a generation. We also demonstrate that several demographic and behavioural phenotypes have effects on brain similarity. Collectively, our results provide a comprehensive picture of neurobiological substrates of parent-child similarity, and demonstrate the usability of our dataset for investigating the neurobiological substrates of intergenerational transmission.
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neuroscience
|
pyrpipe: a python package for RNA-Seq workflows The availability of terabytes of RNA-Seq data and continuous emergence of new analysis tools, enable unprecedented biological insight. However, implementing RNA-Seq analysis pipelines in a reproducible, flexible manner is challenging as data gets bigger and more complex. Thus, there is a pressing requirement for frameworks that allows for fast, efficient, easy-to-manage, and reproducibile analysis. Simple scripting has many challenges and drawbacks. We have developed a python package, python RNA-Seq Pipeliner (pyrpipe) that enables straightforward development of flexible, reproducible and easy-to-debug computational pipelines purely in python, in an object-oriented manner. pyrpipe provides access to popular RNA-Seq tools, within python, via easy-to-use high level APIs. Pipelines can be customized by integrating new python code, third-party programs, or python libraries. Users can create checkpoints in the pipeline or integrate pyrpipe into a workflow management system, thus allowing execution on multiple computing environments. pyrpipe produces detailed analysis, and benchmark reports which can be shared or included in publications. pyrpipe is implemented in python and is compatible with python versions 3.6 and higher. To illustrate the rich functionality of pyrpipe, we provide case studies using RNA-Seq data from GTEx, SARS-CoV-2-infected human cells, and Zea mays. All source code is freely available at https://github.com/urmi-21/pyrpipe; the package can be installed from the source or from PyPI (https://pypi.org/project/pyrpipe). Documentation is available at (http://pyrpipe.rtfd.io).
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bioinformatics
|
Tick infestation probability increases with human recreation and exploratory behaviour in great tits Ticks are parasites that feed on the blood of various vertebrate hosts, including many species of bird. Birds can disperse ticks over short and long distances, therefore impacting tick population dynamics. The likelihood that birds attract ticks should depend on their behaviour and the environment. We studied various key ecological variables (breeding density, human disturbance) and phenotypic traits (exploratory behaviour; body condition) proposed to predict tick burden in great tits (Parus major). Our study spanned over three years and 12 human-recreated plots, equipped with nest-boxes in southern Germany. Adult breeders were assessed for exploratory behaviour, tick burden, and body condition. For each plot, human disturbance was quantified as a human recreational pressure index during biweekly nest box inspections by scoring the number of recreants using the plots. Infestation probability but not tick burden increased with exploratory behaviour. We also found moderate support for a positive effect of recreational pressure on infestation probability. Further, body condition negatively predicted tick burden. Individuals were repeatable in tick burden across years. Our study implies that infestation probability and tick burden are governed by distinct ecological and phenotypic drivers. Our findings also highlight the importance of incorporating ecological and individual variation in host phenotypes to predict spatiotemporal distributions of ticks in nature. (207/250-word limit)
Lay SummaryTicks use many birds as hosts, but why do some individuals have more or fewer ticks? Using a data collected over three years on great tit adults inhabiting 12 different nest-box plots, we showed that more explorative birds and those in highly recreated habitats were more likely to be infested with ticks. Exploratory behaviour and human disturbance could modify great tit habitat choice and, therefore, impact how often a birds and ticks encounter each other.
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ecology
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Is male dimorphism under sexual selection in humans? A meta-analysis Humans are sexually dimorphic: men and women differ in body build and composition, craniofacial structure, and voice pitch, likely mediated in part by developmental testosterone. Sexual selection hypotheses posit that, ancestrally, more masculine men may have acquired more mates and/or sired more viable offspring. Thus far, however, evidence for either association is unclear. Here, we meta-analyze the relationships between six masculine traits and mating/reproductive outcomes (96 studies, 474 effects, N = 177,044). Voice pitch, height, and testosterone all predicted mating; however, strength/muscularity was the strongest and only consistent predictor of both mating and reproduction. Facial masculinity and digit ratios did not significantly predict either. There was no clear evidence for any effects of masculinity on offspring viability. Our findings support arguments that strength/muscularity can be considered sexually selected in humans, but raise concerns over other forms of masculinity and highlight the need to increase tests of evolutionary hypotheses outside of industrialized populations.
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evolutionary biology
|
Evidence accumulation and associated error-related brain activity as computationally informed prospective predictors of substance use in emerging adulthood RationaleSubstance use peaks during the developmental period known as emerging adulthood (ages 18-25), but not every individual who uses substances during this period engages in frequent or problematic use. Although individual differences in neurocognition appear to predict use severity, mechanistic neurocognitive risk factors with clear links to both behavior and neural circuitry have yet to be identified. Here we aim to do so with an approach rooted in computational psychiatry, an emerging field in which formal models are used to identify candidate biobehavioral dimensions that confer risk for psychopathology.
ObjectivesWe test whether lower efficiency of evidence accumulation (EEA), a computationally-characterized individual difference variable that drives performance on the go/no-go and other neurocognitive tasks, is a risk factor for substance use in emerging adults.
Methods and ResultsIn an fMRI substudy within a sociobehavioral longitudinal study (n=106), we find that lower EEA and reductions in a robust neural-level correlate of EEA (error-related activations in salience network structures) measured at ages 18-21 are both prospectively related to greater substance use during ages 22-26, even after adjusting for other well-known risk factors. Results from Bayesian model comparisons corroborated inferences from conventional hypothesis testing and provided evidence that both EEA and its neuroimaging correlates contain unique predictive information about substance use involvement.
ConclusionsThese findings highlight EEA as a computationally-characterized neurocognitive risk factor for substance use during a critical developmental period, with clear links to both neuroimaging measures and well-established formal theories of brain function.
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neuroscience
|
A transcriptomic census reveals that Rbfox contributes to a broad but selective recapitulation of peripheral tissue splicing patterns in the thymus Thymic epithelial cells (TEC) control the selection of a T-cell repertoire reactive to pathogens but tolerant of self. While this process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, have remained largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, we found that mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific micro-exons in mTEC. We did not find unusual numbers of novel transcripts in TEC and show that Aire, the facilitator of promiscuous gene expression, promotes usage of long transcripts but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of non-promiscuously expressed SF genes amongst which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss of function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the Rbfox family, to generate a broad but selective representation of the peripheral splice isoform repertoire.
|
immunology
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Coastal development and human recreation disturbance limit Australian Pied Oystercatcher Haematopus longirostris population sizes on E Australian beaches Australian Pied Oystercatchers Haematopus longirostris and their habitat were surveyed on 72 beaches and 674 km of coastline, from Fraser Island, Queensland to near the New South Wales-Victoria state border, in 2015-2018. A grand total of 232 individual birds (the sum of mean beach counts) and 41 oystercatcher territories were counted. Regression models for mean oystercatcher count density indicated a positive response to the abundance of the surf clam Donax deltoides, a positive New South Wales Far North Coast regional effect and a negative response to the proportion of urban beach. Models for oystercatcher territory density indicated a positive Far North Coast effect and a negative response to pedestrian access density. This report upgrades the coastal development and human recreation disturbance threats for the species.
|
ecology
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Ecological opportunity, radiation events and genomic innovations shaped the episodic evolutionary history of papillomaviruses Papillomaviruses (PVs) have a wide host range, infecting mammals, birds, turtles, and snakes. The recent discovery of PVs in different fish species allows for a more complete reconstruction of the evolutionary history of this viral family. In this study we perform phylogenetic dating to analyse evolutionary events that occurred during PV evolution, as well as to estimate speciation and evolutionary rates.
We have used four different data sets to explore and correct for potential biases that particular taxa combinations may introduce during molecular time inference. When considering the evolution of substitution rates we observed that short-term rate estimates are much higher than long-term rate estimates, also known as the time-dependent rate phenomenon. When considering the evolution of viral branching events (as a proxy for speciation rates), we show that these have not been constant through time, suggesting the occurrence of distinct evolutionary events such as adaptive radiations and/or changes in the available host niches. In a joint analysis with host speciation rates, we identified at least four different evolutionary periods, suggesting that the evolution of PVs has been multiphasic, and thus refining the previously suggested biphasic evolutionary scenario. Thanks to the discovery of novel PVs in basal hosts and to the implementation of a time-dependent rate model for molecular dating, our results provide new insights into the evolutionary history of PVs. In this updated evolutionary scenario, ecological opportunity appears as one main driving force for the different radiation and key-innovation events we observe.
|
evolutionary biology
|
Estimating plasmid conjugation rates: a new computational tool and a critical comparison of methods Plasmids are important vectors for the spread of genes among diverse populations of bacteria. However, there is no standard method to determine the rate at which they spread horizontally via conjugation. Here, we compare commonly used methods on simulated and experimental data, and show that the resulting conjugation rate estimates often depend strongly on the time of measurement, the initial population densities, or the initial ratio of donor to recipient populations. Differences in growth rate, e.g. induced by sub-lethal antibiotic concentrations or temperature, can also significantly bias conjugation rate estimates. We derive a new end-point measure to estimate conjugation rates, which extends the well-known Simonsen method to include the effects of differences in population growth and conjugation rates from donors and transconjugants. We further derive analytical expressions for the parameter range in which these approximations remain valid. We present an easy to use R package and web interface which implement both new and previously existing methods to estimate conjugation rates. The result is a set of tools and guidelines for accurate and comparable measurement of plasmid conjugation rates.
HighlightsO_LIWe present an R package and Shiny app to estimate conjugation rates from experimental data
C_LIO_LIWe highlight the diversity of existing measures used to estimate conjugation rates
C_LIO_LIWe show these measures are biased for populations with differing growth and conjugation rates from donors or transconjugants
C_LIO_LIWe develop a new end-point measure which accounts for such differences in growth and conjugation rates
C_LI
|
microbiology
|
In Vivo Transcriptomic Profiling using Cell Encapsulation Identifies Effector Pathways of Systemic Aging Sustained exposure to a young systemic environment rejuvenates aged organisms and promotes cellular function. However, due to the intrinsic complexity of tissues it remains challenging to pinpoint niche-independent effects of circulating factors on specific cell populations. Here we describe a method for the encapsulation of human and mouse skeletal muscle progenitors in diffusible polyethersulfone hollow fiber capsules that can be used to profile systemic aging in vivo independent of heterogeneous short-range tissue interactions. We observed that circulating long-range signaling factors in the old systemic environment lead to an activation of Myc and E2F transcription factors, induce senescence and suppress myogenic differentiation. Importantly, in vitro profiling using young and old serum in 2D culture does not capture all pathways deregulated in encapsulated cells in aged mice. Thus, in vivo transcriptomic profiling using cell encapsulation allows for the characterization of effector pathways of systemic aging with unparalleled accuracy.
|
bioengineering
|
Identification of X-chromosomal genes that drive global X-dosage effects in embryonic stem cells X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Here, double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signalling pathway and delaying differentiation. To identify the genetic basis of these sex differences, we have performed a series of CRISPR knockout screens in murine embryonic stem cells to comprehensively identify X-linked genes that cause the female pluripotency phenotype. We found multiple genes that act in concert, among which Klhl13 plays a central role. We show that this E3 ubiquitin ligase substrate adaptor protein promotes pluripotency factor expression, delays differentiation and represses MAPK target genes, and we identify putative substrates. We thus elucidate the mechanisms that drive sex-induced differences in pluripotent cells with implications for gender medicine in the context of induced pluripotent stem cell based therapies.
|
genetics
|
Mutagenic effectiveness and efficiency of gamma rays and sodium azide in M2 generation of Cowpea Legumes play a pivotal role in combating the chronic hunger and malnutrition in the developing nations and are also ideal crops to achieve global food and nutrition security. In the era of climate change, erratic rainfalls, depleting arable land and water resource, feeding the rapidly growing population is a challenging task. Like most other pulses, cowpea is a self pollinated, nutritious, versatile and widely adapted grain legume, but harbor a little accessible genetic variability. Lack of sufficient genetic variability and small size of flowers, traditional plant breeding methods are not enough to meet the demands of improvement of cowpea. Hence, induced mutagenesis was employed to induce significant genetic variability across a range of agro-economical traits in two cowpea varieties Gomati VU-89 and Pusa- 578 from M1 to M4 generations. The success of induced mutagenesis largely depends on the selection of appropriate mutagen, its dose, effectiveness and efficiency. Hence present study was conduct to assess the effectiveness and efficiency of single and combined doses of sodium azide and gamma rays to set an appropriate protocol for induced mutagenesis experimentation in cowpea.
|
plant biology
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Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization Identifying protein-protein interactions (PPI) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditional in vivo PPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. This in vivo tool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions in Nicotiana benthamiana leaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.
|
cell biology
|
Oscillatory and aperiodic neural activity jointly predict language learning Memory formation involves the synchronous firing of neurons in task-relevant networks, with recent models postulating that a decrease in low frequency oscillatory activity underlies successful memory encoding and retrieval. However, to date, this relationship has been investigated primarily with face and image stimuli; considerably less is known about the oscillatory correlates of complex rule learning, as in language. Further, recent work has shown that non-oscillatory (1/f) activity is functionally relevant to cognition, yet its interaction with oscillatory activity during complex rule learning remains unknown. Using spectral decomposition and power-law exponent estimation of human EEG data (17 females, 18 males), we show for the first time that 1/f and oscillatory activity jointly influence the learning of word order rules of a miniature artificial language system. Flexible word order rules were associated with a steeper 1/f slope, while fixed word order rules were associated with a shallower slope. We also show that increased theta and alpha power predicts fixed relative to flexible word order rule learning and behavioural performance. Together, these results suggest that 1/f activity plays an important role in higher-order cognition, including language processing, and that grammar learning is modulated by different word order permutations, which manifest in distinct oscillatory profiles.
|
neuroscience
|
Causal relationship between the right auditory cortex and speech-evoked envelope-following response: Evidence from combined tDCS and EEG Speech-evoked envelope-following response (EFR) reflects brain encoding of speech periodicity that serves as a biomarker for pitch and speech perception and various auditory and language disorders. While EFR is thought to originate from the subcortex, recent research illustrated a right-hemispheric cortical contribution to EFR. However, it is unclear whether this contribution is causal. This study aimed to establish this causality by combining transcranial direct current stimulation (tDCS) and measurement of EFR (pre- and post-tDCS) via scalp-recorded electroencephalography (EEG). We applied tDCS over the left and right auditory cortices in right-handed normal-hearing participants and examined whether altering cortical excitability via tDCS causes changes in EFR during monaural listening to speech syllables. We showed significant changes in EFR magnitude when tDCS was applied over the right auditory cortex compared to sham stimulation for the listening ear contralateral to the stimulation site. No such effect was found when tDCS was applied over the left auditory cortex. Crucially, we further observed a hemispheric laterality where after-effect was significantly greater for tDCS applied over the right than the left auditory cortex in the contralateral ear condition. Our finding thus provides the first evidence that validates the causal relationship between the right auditory cortex and EFR.
|
neuroscience
|
Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohns disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohns disease and non-Crohns tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.
|
genomics
|
Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology, as well as accelerate the development of next generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein we demonstrate the use of suspendable microcontainers to sort single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. Our microparticles act as solid supports which facilitate cell attachment, partition uniform aqueous compartments, and capture secreted proteins. Using this platform, we demonstrate high-throughput screening of stably- and transiently-transfected producer cells based on relative IgG production as well as screening of B lymphocytes and hybridomas based on antigen-specific antibody production using commercially available flow sorters. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1,000,000 events in less than an hour. The reported microparticles can be easily stored, and distributed as a consumable reagent amongst researchers, democratizing access to high-throughput functional cell screening.
|
bioengineering
|
Dopamine pathway characterization during the reproductive mode switch in the pea aphid Aphids are major pests of most of the crops worldwide. Such a success is largely explained by the remarkable plasticity of their reproductive mode. They reproduce efficiently by viviparous parthenogenesis during spring and summer generating important damage on crops. At the end of the summer, viviparous parthenogenetic females perceive the photoperiod shortening and transduce this signal to their embryos that change their reproductive fate to produce sexual individuals: oviparous females and males. After mating, those females lay cold-resistant eggs. Earlier studies showed that some transcripts coding for key components of dopamine pathway were regulated between long days and short days conditions suggesting that dopamine might be involved in the transduction of seasonal cues prior to reproductive mode switch. In this study, we aimed at going deeper into the characterization of the expression dynamics of this pathway but also in the analysis of its functional role in this context in the pea aphid Acyrthosiphon pisum. We first analysed the level of expression of ten genes of this pathway in embryos and larval heads of aphids reared under long days (asexual producers) or short days (sexual producers) conditions. We then performed in situ hybridization experiments to localize in embryos the ddc and pale transcripts that are coding for two key enzymes in dopamine synthesis. Finally, Using CRISPR-Cas9 mutagenesis in eggs produced after the mating of sexual individuals, we targeted the ddc gene. We could observe strong melanization defaults in ddc mutated eggs, which confidently mimicked the Drosophila ddc phenotype. Nevertheless, such a lethal phenotype did not allow us to validate the involvement of dopamine as a signaling pathway necessary to trigger the reproductive mode switch in embryos.
|
developmental biology
|
Heat treatment functionalizes hepatocyte-like cells derived from human embryonic stem cells. Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. Although tremendous efforts have been made to facilitate hepatic functions of hPSC-HLCs using growth factors and chemicals, these cells have not yet reached hepatic functions comparable to hepatocytes in vivo. Therefore, there exists a critical need to use an alternative trigger to facilitate hepatic functions in hPSC-HLCs. We noted that human liver temperature (around 39{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), yet hepatocytes are generally cultured at 37{degrees}C in-vitro. Here we showed that hepatic functions of hPSC-HLCs would be facilitated under physiological liver temperatures. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39{degrees}C and 42{degrees}C. 42{degrees}C-treatment caused significantly greater cell death compared to 39{degrees}C. We also confirmed the increases of hepatic functions, such as secretion of albumin, cytochrome P450 3A4 (CYP3A4) activities, and collagen productions, without severe cell damages. To elucidate the underlying mechanisms of heat-induced hepatic functions, RNA-seq was to identify gene expression signatures due to 39{degrees}C-treated hESC-HLCs. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs, and lead to the realization of drug discovery efforts and drug toxicological tests.
Significance statementHepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. We noted that human liver temperature (around 39{degrees}C) is higher than normal human body temperature (around 36.5{degrees}C), affecting the in-vitro hepatic functions of hPSC-HLCs, such as metabolic activities. Here we showed that hepatic functions of hPSC-HLCs, albumin secretion, CYP3A4 activities, and collagen production would be facilitated under physiological liver temperatures at 39{degrees}C, without severe cell damages. RNA-seq was used to elucidate the underlying mechanisms of heat-induced hepatic functions. This study also showed the possible mechanisms of heat-induced hepatic function via glucocorticoid receptor pathway and molecular chaperons.
|
bioengineering
|
Assortative mate choice and epistatic mating-trait architecture induce complex movement of the crow hybrid zone Hybrid zones provide a window into the evolutionary processes governing species divergence. While the role of postzygotic isolation has been extensively characterized in the context of hybrid zones, the contribution of prezygotic isolation is less well explored. Here, we investigate the effects of assortative mate choice, the underlying preference function and mating-trait architecture, and the strength of sexual selection on hybrid zone dynamics. We explore this question by means of a mathematical model parameterized with phenotype and genotype data from the hybrid zone between all-black carrion and grey-coated hooded crows. The best-fit model resulted in narrow clines for two mating-trait loci coding for colour phenotype maintained by a moderate degree of assortative mating. Epistasis between the two loci induced hybrid-zone movement in favor of alleles conveying dark plumage followed by a shift in the opposite direction favouring grey-coated phenotypes [~]1,200 generations after secondary contact. Unlinked neutral loci diffused near-unimpeded across the zone. These results were generally robust to the choice of matching rule (self-referencing or parental imprinting) and effects of genetic drift. Overall, this study illustrates under which conditions assortative mating can maintain steep clines in mating-trait loci without generalizing to genome-wide reproductive isolation. It further emphasizes the importance of mating-trait architecture for spatio-temporal hybrid-zone dynamics.
|
evolutionary biology
|
Contributions of the four essential entry glycoproteins to HSV-1 tropism and the selection of entry routes. Herpes Simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell-type dependent entry routes is unknown. To begin addressing this, we previously pseudotyped VSV, lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSV{Delta}G-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, a cellular receptor, and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSV{Delta}G-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein gC (VSV{Delta}G-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner.
ImportanceHerpes simplex viruses (HSV-1 and HSV-2) contain up to 16 different proteins in their envelopes. Four of these, glycoproteins gB, gD, gH, and gL, are termed essential with regard to entry whereas the rest are typically referred to as non-essential based on the entry phenotypes of the respective single genetic deletions. However, the single-gene deletion approach, which relies on robust loss-of-function phenotypes, may be confounded by functional redundancies among the many HSV-1 envelope proteins. We have developed a pseudotyping platform, in which the essential four entry glycoproteins are isolated from the rest, which can be added back individually for systematic gain-of-function entry experiments. Here, we show the utility of this platform for dissecting the contributions of HSV envelope proteins, both the essential four and the remaining dozen (using gC as an example), to HSV entry.
|
microbiology
|
Coevolutionary dynamics of genetic traits and their long-term extended effects under non-random interactions Organisms continuously modify their living conditions via extended genetic effects on their envi-ronment, microbiome, and in some species culture. These effects can impact the fitness of current but also future conspecifics due to non-genetic transmission via ecological or cultural inheritance. In this case, selection on a gene with extended effects depends on the degree to which current and future genetic relatives are exposed to modified conditions. Here, we detail the selection gradient on a quantitative trait with extended effects in a patch-structured population, when gene flow between patches is limited and ecological inheritance within patches can be biased towards offspring. Such a situation is relevant to understand evolutionary driven changes in individual condition that can be preferentially transmitted from parent to offspring, such as cellular state, micro-environments (e.g., nests), pathogens, microbiome, or culture. Our analysis quantifies how the interaction between limited gene flow and biased ecological inheritance influences the joint evolutionary dynamics of traits together with the conditions they modify, helping understand adaptation via non-genetic modifications. As an illustration, we apply our analysis to a gene-culture coevolution scenario in which genetically-determined learning strategies coevolve with adaptive knowledge. In particular, we show that when social learning is synergistic, selection can favour strategies that generate remarkable levels of knowledge under intermediate levels of both vertical cultural transmission and limited dispersal. More broadly, our theory yields insights into the interplay between genetic and non-genetic inheritance, with implications for how organisms evolve to transform their environments.
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evolutionary biology
|
Conditional repeatability and the variance explained by reaction norm variation in random slope models Individuals differ in average phenotypes and in sensitivity to environmental variation. Such context-sensitivity can be modelled as random-slope variation. Random-slope variation implies that the proportion of between-individual variation varies across the range of a covariate (environment/context/time/age) and has thus been called conditional repeatability. We propose to put conditional repeatabilities in perspective of the total phenotypic variance and suggest a way of standardization using the random-slope coefficient of determination [Formula]. Furthermore, we illustrate that the marginalized repeatability Rmar averaged across an environmental gradient offers a biologically relevant description of between-individual variation. We provide simple equations for calculating key descriptors of conditional repeatabilities, clarify the difference between random-intercept variation and average between-individual variation and make recommendations for comprehensive reporting. While we introduce the concept with individual variation in mind, the framework is equally applicable to other type of between-group/cluster variation that varies across some (environmental) gradient.
|
animal behavior and cognition
|
Filamentous Bacteriophage Delay Healing of Pseudomonas-Infected Wounds We have identified a novel role for filamentous bacteriophage in the delayed healing associated with chronic Pseudomonas aeruginosa (Pa) wound infections. Pf phage delays wound re-epithelialization in the absence of live Pa, indicative that Pf effects on wound healing are independent of Pa pathogenesis. Pf phage directly inhibits autocrine signaling of CXCL1 (KC) to impede keratinocyte migration and wound re-epithelization. In agreement with these studies, a prospective cohort study of 36 human patients with chronic Pa wound infections revealed that wounds infected with Pf positive strains of Pa took longer to heal and were more likely to increase in size compared to wounds infected with Pf negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian target cells. We propose that Pf phage may have potential as a biomarker and therapeutic target in delayed wound healing.
|
microbiology
|
Automated Annotation of Cell Identities in Dense Cellular Images Although identifying cell names in dense image stacks is critical in analyzing functional whole-brain data enabling comparison across experiments, unbiased identification is very difficult, and relies heavily on researchers experiences. Here we present a probabilistic-graphical-model framework, CRF_ID, based on Conditional Random Fields, for unbiased and automated cell identification. CRF_ID focuses on maximizing intrinsic similarity between shapes. Compared to existing methods, CRF_ID achieves higher accuracy on simulated and ground-truth experimental datasets, and better robustness against challenging noise conditions common in experimental data. CRF_ID can further boost accuracy by building atlases from annotated data in highly computationally efficient manner, and by easily adding new features (e.g. from new strains). We demonstrate cell annotation in C. elegans images across strains, animal orientations, and tasks including gene-expression localization, multi-cellular and whole-brain functional imaging experiments. Together, these successes demonstrate that unbiased cell annotation can facilitate biological discovery, and this approach may be valuable to annotation tasks for other systems.
|
neuroscience
|
Glucose-dependent activation, activity, and deactivation of beta cell networks in acute mouse pancreas tissue slices Many details of glucose-stimulated intracellular calcium changes in beta cells during activation, activity and deactivation, as well as their concentration-dependence, remain to be described. Classical physiological experiments indicated that in islets functional differences between individual cells are largely attenuated, but recent findings suggest considerable intercellular heterogeneity, with some cells possibly coordinating the collective responses. To address the above with an emphasis on heterogeneity and describing the relations between classical physiological and functional network properties, we performed functional multicellular calcium imaging in mouse pancreas tissue slices over a wide range of glucose concentrations. During activation, delays to activation of cells and first responder-any cell delays shortened, and the sizes of simultaneously responding clusters increased with increasing glucose. Exactly the opposite characterized deactivation. The frequency of fast calcium oscillations during activity increased with increasing glucose up to 12 mM glucose, beyond which oscillation duration became longer, resulting in a homogenous increase in active time. In terms of functional connectivity, islets progressed from a very segregated network to a single large functional unit with increasing glucose. A comparison between classical physiological and network parameters revealed that the first-responders during activation had longer active times during plateau and the most active cells during the plateau tended to deactivate later. Cells with the most functional connections tended to activate sooner, have longer active times, and deactivate later. Our findings provide a common ground for recent differing views on beta cell heterogeneity and an important baseline for future studies of stimulus-secretion and intercellular coupling.
|
physiology
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Molecular mechanism of the pH-dependent calcium affinity in langerin The C-type lectin receptor langerin plays a vital role in the mammalian defense against invading pathogens. Its function hinges on the affinity to its co-factor Ca2+ which in turn is regulated by the pH. We studied the structural consequences of protonating the allosteric pH-sensor histidine H294 by molecular dynamics simulations (total simulation time: about 120 {micro}s) and Markov models. We discovered a mechanism in which the signal that the pH has dropped is transferred to the Ca2+-binding site without transferring the initial proton. Instead, protonation of H294 unlocks a conformation in which a protonated lysine side-chain forms a hydrogen bond with a Ca2+-coordinating aspartic acid. This destabilizes Ca2+ in the binding pocket, which we probed by steered molecular dynamics. After Ca2+-release, the proton is likely transferred to the aspartic acid and stabilized by a dyad with a nearby glutamic acid, triggering a conformational transition and thus preventing Ca2+-rebinding.
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molecular biology
|
The 16S microbiota of Budu, the Malaysian fermented anchovy sauce Budu is a Malaysian fermented anchovy sauce produced by immersing small fishes into a brine solution for 6 to 18 months. Fermentation of the anchovy sauce is contributed partly by microbial enzymes, but little is known about the microbial community in Budu. Therefore, a better understanding of the Budu microbiome is necessary to better control the quality, consistency and safety of the product. In this study, we collected 60 samples from twenty bottles of Budu produced by seven different manufacturers. We analyzed their microbiota based on V3-V4 16S rRNA amplicon sequencing at the time of opening the bottle as well as 3- and 7-months post-opening. Tetragenococcus was the dominant genus in many samples, reaching a maximum proportion of 98.62%, but was found in low abundance, or absent, in other samples. When Budu samples were not dominated by a dominant taxa, we observed a wider genera diversity such as Staphylococcus, Acinetobacter, Halanaerobium and Bacillus. While the taxonomic composition was relatively stable across sampling periods, samples from two brands showed a sudden increase in relative abundance of the genus Chromobacterium in the 7th month. Based on prediction of metagenome functions, non-Tetragenococcus-dominated samples were predicted to have enriched functional pathways related to amino acid metabolism and purine metabolism compared to Tetragenococcus-dominated microbiome; these two pathways are fundamental fermented quality and health attributes of fish sauce. Within the non-Tetragenococcus-dominated group, contributions towards amino acid metabolism and purine metabolism were biased towards the dominant taxa when species evenness is low, while in samples with higher species evenness, the contributions towards the two pathways were predicted to be evenly distributed between taxa.
|
molecular biology
|
Age-related trajectories of DNA methylation network markers: a parenclitic network approach to a family-based cohort of patients with Down Syndrome Despite the cause of Down Syndrome (DS) is well established, the underlying molecular mechanisms that contribute to the syndrome and to the phenotype of accelerated ageing remain largely unknown. DNA methylation profiles are largely altered in DS but it remains unclear how different methylation regions and probes are structured into a network of interactions. We develop and generalize the Parenclitic Networks approach that enables to find correlations between distant CpG probes (and not pronounced as stand-alone biomarkers) and quantifies hidden network changes in DNA methylation. DS and a family-based cohort (including healthy siblings and mothers of persons with DS) is used as a case study. Following this approach, we constructed parenclitic networks and obtained different signatures informative for (i) the differences between individuals with DS from healthy individuals; (ii) differences between young and old healthy individuals and determining the place of DS individuals on this scale; (iii) differences between DS individuals from their age-matched siblings, and (iv) the difference between DS and the adult population (their mothers). Conducted Gene Ontology analysis showed that the CpG network approach is more powerful than the single CpG approach in identifying biological processes related to DS phenotype, like those involved in central nervous system, skeletal muscles, disorders in carbohydrate metabolism, cardiopathology, and oncogenes. Our open-source software implementation is accessible to all researchers. The software includes a complete workflow to construct Parenclitic Networks with any machine learning algorithm as a kernel to build edges. We anticipate a broad applicability of the approach to other diseases.
|
bioinformatics
|
A Generalized Robust Allele-based Genetic Association Test The allele-based association test, comparing allele frequency difference between case and control groups, is locally most powerful. However, application of the classical allelic test is limited in practice, because the method is sensitive to the Hardy-Weinberg equilibrium (HWE) assumption, not applicable to continuous traits, and not easy to account for covariate effect or sample correlation. To develop a generalized robust allelic test, we propose a new allele-based regression model with individual allele as the response variable. We show that the score test statistic derived from this robust and unifying regression framework contains a correction factor that explicitly adjusts for potential departure from HWE, and encompasses the classical allelic test as a special case. When the trait of interest is continuous, the corresponding allelic test evaluates a weighted difference between individual-level allele frequency estimate and sample estimate where the weight is proportional to an individuals trait value, and the test remains valid under Y - dependent sampling. Finally, the proposed allele-based method can analyze multiple (continuous or binary) phenotypes simultaneously and multi-allelic genetic markers, while accounting for covariate effect, sample correlation and population heterogeneity. To support our analytical findings, we provide empirical evidence from both simulation and application studies.
|
genetics
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TrkB agonist antibody delivery to the brain using a TfR1 specific BBB shuttle provides neuroprotection in a mouse model of Parkinson's Disease Single domain shark antibodies that bind to the transferrin receptor 1 (TfR1) on brain endothelial cells can be used to shuttle antibodies and other cargos across the blood brain barrier (BBB). We have fused one of these (TXB4) to differing regions of TrkB and TrkC neurotrophin receptor agonist antibodies (AAb) and determined the effect on agonist activity, brain accumulation and engagement with neurons in the mouse brain following systemic administration. The TXB4-TrkB and TXB4-TrkC fusion proteins retain agonist activity at their respective neurotrophin receptors and in contrast to their parental AAb they rapidly accumulate in the brain reaching nM levels following a single IV injection. Following SC administration, the most active TrkB fusion protein, TXB4-TrkB1, associates with and activates ERK1/2 signalling in TrkB positive cells in the cortex and tyrosine hydroxylase (TH) positive dopaminergic neurons in the substantia nigra pars compacta (SNc). When tested in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinsons disease (PD) TXB4-TrkB1, but not the parental TrkB AAb or a TXB4-TrkC1 fusion protein, completely prevented the 6-OHDA induced death of TH positive neurons in the SNc. In conclusion, the fusion of the TXB4 TfR1 binding module allows a TrkB AAb to reach neuroprotective concentrations in the brain parenchyma following systemic administration.
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neuroscience
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A Combined Evidence Approach to Prioritize Nipah Virus Inhibitors Nipah Virus (NiV) came into limelight due to an outbreak in Kerala, India. NiV causes severe disease and death in people with over 75% case fatality rate. It is a public health concern and has the potential to become a global pandemic. Lack of treatment has forced the containment methods to be restricted to isolation and surveillance. WHOs R&D Blueprint list of priority diseases (2018) indicates that there is an urgent need for accelerated research & development for addressing NiV. In the quest for druglike NiV inhibitors (NVIs) a thorough literature search followed by systematic data curation was conducted. Rigorous data analysis was done with curated NVIs for prioritizing druglike compounds. For the same, more than 1800 descriptors of NVIs were computed and comparative analysis was performed with the FDA approved small molecules and antivirals. These compounds were further evaluated through PAINS filter to study their toxicity profile. Simultaneously, compounds were also prioritized based on the robustness of the assays through which they were identified. Our efforts lead to the creation of a well-curated structured knowledgebase of 182 NVIs with 98 small molecule inhibitors. The reported IC50/EC50 values for some of these inhibitors are in the nanomolar range - as low as 0.47 nM. In order to prioritize these inhibitors, we performed several tests and applied filters to identify drug-like non-toxic compounds. Of 98, a few compounds passed DruLito & PAINS filters exhibiting drug-like properties and were also prioritized in an independent screen based only the assay robustness. The NVIs have diverse structural features and offer a wide spectrum of ways in which they can be developed further as druglike molecules. We report a knowledgebase for furthering the development of NVIs. The platform has a diverse set of 98 NVIs of which a few have been prioritized based on a combined evidence strategy. The platform has the provision to submit new inhibitors as and when reported by the community for further enhancement of NiV inhibitor landscape.
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bioinformatics
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