Modulation of P2X4 pore closure by magnesium,potassium, and ATP The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 {micro}s we have investigated how metal-complexed ATP stabilizes the channels open state and prevents its closing. We have identified two metal-binding sites, magnesium (Mg2+) and potassium (K+), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 resisted collapsing of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and we found evidence of pore collapse. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+.
Robust and Remote Measurement of Heart Rate Based on A Surveillance Camera Arrhythmia is a marked symptom of many cardiovascular diseases (CVDs). The accurate and timely detection of heart rate can greatly reduce the harm of arrhythmia to people. However, it is still a challenge to robustly and remotely measure heart rate in daily life due to the changing environmental conditions during measurement, such as the varying light intensity, the movement of people, and the uncertain distance between the sensor to people. In this study, we propose a method to accurately measure human heart rate within a distance of 4.5 meters under different light intensities by simply using a surveillance camera. After a 20-second color video of a persons hand is captured by the camera, a method based on the Fast Fourier Transform (FFT) algorithm is designed to extract the blood volume pulse wave to calculate the heart rate. According to the comparison between the real heart rate and results measured by electrocardiography (ECG), the proposed method achieves an accuracy of 95.8% when the measurement is performed within a distance of 4.5 meters and 90% when within 5.0 meters. Our experiments show that when the illuminance varies between 100-1000 lux (lighting level indoor), we still get the correct results. Our experiments also demonstrate that the proposed method accurately obtain heart rate even when the light intensity is below 32 lux (300-500 lux in a workplace environment). The methods strong adaptability to changing environmental conditions makes it applicable to many scenarios, such as homes of the elderly, classrooms, and other public spaces.
Many ways to stick the landing: novel righting strategies allow spotted lanternfly nymphs to land on diverse substrates Unlike large animals, insects and other very small animals are so unsusceptible to impact-related injuries that they can use falling for dispersal and predator evasion. Reorienting to land upright can mitigate lost access to resources and predation risk. Such behaviors are critical for the spotted lanternfly (SLF) (Lycorma delicatula), an invasive, destructive insect pest spreading rapidly in the US. High-speed video of SLF nymphs released under different conditions showed that these insects self-right using both active midair righting motions previously reported for other insects, and novel post-impact mechanisms that take advantage of their ability to experience near-total energy loss on impact. Unlike during terrestrial self-righting, in which an animal initially at rest on its back uses appendage motions to flip over, SLF nymphs impacted the surface at varying angles and then self-righted during the rebound using coordinated body rotations, foot-substrate adhesion, and active leg motions. These previously-unreported strategies were found to promote disproportionately upright, secure landings on both hard, flat surfaces and tilted, compliant host plant leaves. Our results highlight the importance of examining biomechanical phenomena in ecologically-relevant contexts, and show that, for small animals, the post-impact bounce period can be critical for achieving an upright landing. FGF8-mediated signaling regulates tooth developmental pace during odontogenesis The developing human and mouse teeth constitute an ideal model system to study the regulatory mechanism underlying organ growth control due to the fact that their teeth share highly conserved and well-characterized developmental processes and their developmental tempo vary notably. In the current study, we manipulated heterogenous recombination between human and mouse dental tissues and demonstrate that the dental mesenchyme dominates the tooth developmental tempo and FGF8 could be a critical player during this developmental process. Forced activation of FGF8 signaling in the dental mesenchyme of mice promoted cell proliferation, prevented cell apoptosis via p38 and perhaps PI3K-Akt intracellular signaling, and impelled the transition of the cell cycle from G1- to S-phase in the tooth germ, resulting in the slowdown of the tooth developmental pace. Our results provide compelling evidence that extrinsic signals can profoundly affect tooth developmental tempo and the dental mesenchymal FGF8 could be a pivotal factor in controlling developmental pace in a non-cell-autonomous manner during mammalian odontogenesis. HaloTag9: an engineered protein tag to improve fluorophore performance HaloTag9 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag9 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag9 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging. Natural variations in reproductive timing of brood size and X-chromosome nondisjunction in Caenorhabditis elegans Reproductive timing underlies self-sperm production and X-chromosome nondisjunction rate in Caenorhabditis elegans. These traits can be measured as brood size and male-production rate, and brood size decreases and male-production rate increases as worms age. These phenotypic changes occur simultaneously; however, whether these changes share common underlying genetic architectures still remains unclear. To enhance our understanding of reproductive timing in C. elegans, we measured and compared these two reproductive timing-related traits of 96 wild strains during early, late and total reproductive periods. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. The brood size and the male-production rate were not genetically correlated, implying that they might have different genetic architectures and that meiosis quality can be maintained despite increased progeny-production in the late reproductive period caused by more self-sperm. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life history traits in wild C. elegans strains. SARS-CoV-2 infection induces soluble platelet activation markers and PAI-1 in the early moderate stage of COVID-19 IntroductionCoagulation dysfunction and thromboembolism emerge as strong comorbidity factors in severe COVID-19. However, it is unclear when particularly platelet activation markers and coagulation factors dysregulated during the pathogenesis of COVID-19. Here, we sought to assess the levels of coagulation and platelet activation markers at moderate and severe stages of COVID-19 to understand the pathogenesis.

MethodsTo understand this, hospitalized COVID-19 patients with (severe cases that required intensive care) or without pneumonia (moderate cases) were recruited. Phenotypic and molecular characterizations were performed employing basic coagulation tests including PT, APTT, D-Dimer and TFPI. The flow cytometry-based multiplex assays were performed to assess FXI, anti-thrombin, prothrombin, fibrinogen, FXIII, P-selectin, sCD40L, plasminogen, tissue-plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-Dimer.

ResultsThe investigations revealed induction of plasma P-selectin and CD40 ligand (sCD40L) in moderate COVID-19 cases which were significantly abolished with the progression of COVID-19 severity. Moreover, a profound reduction in plasma tissue factor pathway inhibitor (TFPI) and FXIII were identified particularly in the severe COVID-19. Further analysis revealed fibrinogen induction in both moderate and severe patients. Interestingly, an elevated PAI-1 more prominently in moderate, and tPA particularly in severe COVID-19 cases were observed. Particularly, the levels of fibrinogen and tPA directly correlated with the severity of the disease.

ConclusionsIn summary, induction of soluble P-selectin, sCD40L, fibrinogen and PAI-1 in moderate COVID-19 cases suggests the activation of platelets and coagulation system before patients require intensive care. These findings would help in designing better thromboprophylaxis to limit the COVID-19 severity. De novo identification of maximally deregulated subnetworks based on multi-omics data with DeRegNet BackgroundWith a growing amount of (multi-)omics data being available, the extraction of knowledge from these datasets is still a difficult problem. Classical enrichment-style analyses require predefined pathways or gene sets that are tested for significant deregulation to assess whether the pathway is functionally involved in the biological process under study. De novo identification of these pathways can reduce the bias inherent in predefined pathways or gene sets. At the same time, the definition and efficient identification of these pathways de novo from large biological networks is a challenging problem.

ResultsWe present a novel algorithm, DeRegNet, for the identification of maximally deregulated subnetworks on directed graphs based on deregulation scores derived from (multi-)omics data. DeRegNet can be interpreted as maximum likelihood estimation given a certain probabilistic model for de-novo subgraph identification. We use fractional integer programming to solve the resulting combinatorial optimization problem. We can show that the approach outperforms related algorithms on simulated data with known ground truths. On a publicly available liver cancer dataset we can show that DeRegNet can identify biologically meaningful subgraphs suitable for patient stratification. DeRegNet is freely available as open-source software.

ConclusionThe proposed algorithmic framework and its available implementation can serve as a valuable heuristic hypothesis generation tool contextualizing omics data within biomolecular networks. Trypanosomal variant surface glycoprotein expression in human African trypanosomiasis patients Trypanosoma brucei gambiense, an extracellular protozoan parasite, is the primary causative agent of human African trypanosomiasis. T. b. gambiense is endemic to West and Central Africa, where it is transmitted by the bite of infected tsetse flies. In the bloodstream of an infected host, the parasite evades antibody recognition by altering the Variant Surface Glycoprotein (VSG) that forms a dense coat on its cell surface through a process known as antigenic variation. Each VSG has a variable N-terminal domain exposed to the host and a less variable C-terminal domain that is partially hidden from host antibodies. Our lab developed VSG-seq, a targeted RNA-seq method, to study VSG gene expression in T. brucei. Studies using VSG-seq to characterize antigenic variation in a mouse model have revealed marked diversity in VSG expression within parasite populations, but this finding has not yet been validated in a natural human infection. Here, we used VSG-seq to analyze VSGs expressed in the blood of 12 patients infected with T. b. gambiense. The number of VSGs identified per patient ranged from one to 14; notably, two VSGs were shared by more than one patient. Analysis of expressed VSG N-terminal domain types revealed that 82% of expressed VSGs encoded a type B N-terminus, a bias not seen in datasets from other T. brucei subspecies. C-terminal types in T. b. gambiense infection were also restricted. These results demonstrate a bias either in the underlying VSG repertoire of T. b. gambiense or in this subspecies expression of VSGs during infection. This work demonstrates the feasibility of using VSG-seq to study antigenic variation in human infections and highlights the importance of understanding VSG repertoires in the field.

Author SummaryHuman African Trypanosomiasis is a neglected tropical disease primarily caused by the extracellular parasite Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their dense surface coat of Variant Surface Glycoprotein (VSG). Despite the important role of VSGs in prolonging infection, VSG expression during natural human infections is poorly understood. A better understanding of natural VSG gene expression dynamics can clarify the mechanisms that T. brucei uses to alter its VSG coat and improve how trypanosomiasis is diagnosed in humans. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that parasites in both natural and experimental infections express diverse sets of VSGs. Design, Mutate, Screen: High-throughput creation of genetic clocks with different period-amplitude characteristics Directed evolution has become an invaluable tool in protein engineering and has also greatly influenced the construction of synthetic gene circuits. The ability to generate diversity at precise targets for directed evolution approaches has improved vastly, allowing researchers to create large, specific mutant libraries with relative ease. Screening approaches for large mutant libraries have similarly come a long way, especially when the desired behavior can easily be tested for with static, single time-point assays. For more complex gene circuits with dynamic phenotypes that change over time, directed evolution approaches to controlling and tuning circuit behavior have been hindered by the lack of sufficiently high-throughput screening methods to isolate variants with desired characteristics. Here we utilize directed mutagenesis and multiplexed microfluidics to develop a workflow for creating, screening and tuning dynamic gene circuits that operate at the population level. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and tune its dynamics while uncovering principles regarding its behavior. Lastly, we utilize this directed evolution workflow to construct a new synchronized genetic oscillator that exhibits robust dynamics over long time scales. DCIS genomic signatures define biology and correlate with clinical outcome: a Human Tumor Atlas Network (HTAN) analysis of TBCRC 038 and RAHBT cohorts Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We made observations on DNA, RNA, and protein expression, and generated a de novo clustering scheme for DCIS that represents a fundamental transcriptomic organization at this early stage of breast neoplasia. Distinct stromal expression patterns and immune cell compositions were identified. We found RNA expression patterns that correlate with later events. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/448585v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1755117org.highwire.dtl.DTLVardef@11ebdc0org.highwire.dtl.DTLVardef@13ba799org.highwire.dtl.DTLVardef@1784502_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LINew transcriptomic classification solution reveals 3 major subgroups in DCIS.
C_LIO_LIFour stroma-specific signatures identified.
C_LIO_LIutcome analysis identifies pathways involved in DCIS progression.
C_LIO_LICNAs characterize high risk of distant relapse IBC subtypes observed in DCIS.
C_LI The Tabula Sapiens: a multiple organ single cell transcriptomic atlas of humans Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual. Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific pre-hydrogel digests. ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities which were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissuespecific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. ECM conditioned media had only a minor effect on upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, the extracellular matrix protein type XXII collagen had similar expression patterns as paxillin, with smaller effect sizes. Using tissue specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscletendon junctions to study the expression of MTJ specific proteins.

Impact StatementThe muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, it is overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction specific features and markers that should be included in tissue models of the muscletendon unit, while gaining a deeper understanding of the natural junction. This research aims to inform future methods to engineer a more relevant multi-tissue platform to study the muscletendon unit. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Morphology of migration: Associations between wing, and bill morphology and migration in kingbirds (Tyrannus) Morphology is closely linked to locomotion and diet in animals. In animals that undertake long-distance migrations, limb-morphology is under selection to maximize mobility and minimize energy expenditure. Migratory behaviors also interact with diet, such that migratory animals tend to be dietary generalists, while sedentary taxa tend to be dietary specialists. Despite a hypothesized link between migration status and morphology, phylogenetic comparative studies have yielded conflicting findings. We tested for evolutionary associations between migratory status and limb and bill morphology across kingbirds, a pan-American genus of birds with migratory, partially migratory, and sedentary taxa. Migratory kingbirds had longer wings, in agreement with expectations if selection favors improved aerodynamics for long-distance migration. We also found an association between migratory status and bill shape, such that more migratory taxa had wider, deeper, and shorter bills compared to sedentary taxa. However, there was no difference in intraspecific morphological variation among migrants, partial migrants, and residents, suggesting that dietary specialization has evolved independently of migration strategy. The evolutionary links between migration, diet, and morphology in kingbirds uncovered here further strengthen ecomorphological associations that underlie long-distance seasonal movements in animals. Long term analysis of social structure: evidence of age-based consistent associations in male Alpine ibex Despite its recognized importance for understanding the evolution of animal sociality as well as for conservation, long term analysis of social networks of animal populations is still relatively uncommon. We investigated social network dynamics in males of a gregarious mountain ungulate (Alpine ibex, Capra ibex) over ten years focusing on groups, sub-groups and individuals, exploring the dynamics of sociality over different scales. Despite the social structure changing between seasons, the Alpine ibex population was highly cohesive: fission-fusion dynamics lead almost every male in the population to associate with each other male at least once. Nevertheless, we found that male Alpine ibex showed preferential associations that were maintained across seasons and years. Age seemed to be the most important factor driving preferential associations while other characteristics, such as social status, appeared less crucial. We also found that centrality measures were influenced by age and were also related to individual physical condition. The multi-scale and long-term frame of our study helped us show that ecological constrains, such as resource availability, may play a role in shaping associations in a gregarious species, but they cannot solely explain sociality and preferential association that are likely also to be driven by life-history linked physiological and social needs. Our results highlight the importance of long-term studies based on individually recognizable subjects to help us build on our understanding of the evolution of animal sociality. Thirteen independent genetic loci associated with preserved processing speed in a study of cognitive resilience in 330,097 individuals in the UK Biobank Cognitive resilience is the ability to withstand the negative effects of stress on cognitive functioning and is important for maintaining quality of life while aging. UK Biobank (UKB) does not have direct measurements of the same cognitive phenotype at distal timepoints. Therefore, we used number of education years (EY) as a proxy phenotype for past cognitive performance. Current cognitive performance was determined based on processing speed. This approach captured an average time span of 40 years between past and current cognitive performance in 330,097 individuals. A confounding factor was that EY is highly polygenic and masked the genetics of resilience. To overcome this, we employed Genomics Structural Equation Modelling (GenomicSEM) to perform a GWAS-by-subtraction using two GWAS, one GWAS of EY and resilience and a second GWAS of EY but not resilience. Subtracting one from the other generated a GWAS of Resilience. Replication of this approach was shown using independent discovery and replication samples within UKB and the full UKB GWAS results show significant genetic correlation with a GWAS of cognitive change in the independent Health and Retirement Study (N=9,526; P=1.5x10-3). We found 13 independent genetic loci for Resilience. Functional analyses showed enrichment in several brain regions and involvement of specific cell types, including GABAergic neurons (P=6.59x10-8) and glutamatergic neurons (P=6.98x10-6) in the cortex. Gene-set analyses implicated the biological process "neuron differentiation" (P=9.7x10-7) and the cellular component "synaptic part" (P=2.14x10-6). Mendelian randomization analysis showed a causative effect of white matter volume on cognitive resilience. These results enhance neurobiological understanding of resilience. A pipeline to detect the relationship between transposable elements and adjacent genes in host genomes AO_SCPLOWBSTRACTC_SCPLOWUnderstanding the relationship between transposable elements (TEs) and their closest positional genes in the host genome is a key point to explore their potential role in genome evolution. Transposable elements can regulate and affect gene expression not only because of their mobility within the genome but also because of their transcriptional activity. A comprehensive knowledge of structural organization between transposable elements and neighboring genes is important to study TE functional role in gene regulation. We implemented a pipeline to display the relationship between the distribution of TEs and adjacent genes in host genomes. Our tool is freely available here: https://github.com/marieBvr/TEs_genes_relationship_pipeline. Propionate ameliorates diabetes-induced neurological dysfunction through regulating the PI3K/Akt/eNOS signaling pathway A large body of research has established diabetes-related cognitive deterioration, sometimes known as "diabetic encephalopathy." Current evidence supports that oxidative stress, neuronal apoptosis, and cerebral microcirculation weakness are associated with cognition deficits induced by diabetes. The present study explores the effect of propionate on neurological deficits, cerebral blood flow, and oxidative stress in diabetic mice. Propionate can markedly improve neurological function, which is correlated with its capabilities of stimulating nitric oxide (NO) production, increasing cerebral microcirculation, suppressing oxidative stress and reducing neuron loss in the hippocampus. In addition, the results of Western Blotting indicated that the brain-protective function of propionate in streptozocin (STZ)-induced insulin-dependent diabetes mellitus (IDDM) mice is related to phosphoinositide 3-kinase (PI3K)/serine-threonine protein kinase (Akt)/endothelial nitric oxide synthase(eNOS) signaling pathway. In a diabetic mouse model, propionate reduces cerebral microcirculation, hippocampus apoptosis, and neurological impairment. Thus, propionate, now employed as a food preservative, may also help slow diabetes-induced cognitive loss.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/464493v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@81fa09org.highwire.dtl.DTLVardef@1469451org.highwire.dtl.DTLVardef@b37fa1org.highwire.dtl.DTLVardef@8037e7_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIPropionate ameliorates diabetic-induced depression-like behavior, spatial learning and memory deficits.
C_LIO_LIPropionate increases cerebral microcirculation by enhancing NO production in diabetic mice.
C_LIO_LIPropionate suppresses oxidative stress and reduces neuron loss in the hippocampus.
C_LIO_LIThe brain-protective function of propionate is related to PI3K/Akt/eNOS signaling pathway.
C_LI A nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an olfactory chemoattractant for the grass puffer Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this unknown function TTX analog as an olfactory chemoattractant. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. Plasticity of the dopaminergic phenotype and of locomotion in larval zebrafish induced by changes in brain excitability during the embryonic period During the embryonic period, neuronal communication starts before the establishment of the synapses with forms of neuronal excitability, called here Embryonic Neuronal Excitability (ENE). ENE has been shown to modulate the correct unfolding of development transcriptional programs but the global consequences for the developing organisms are not all understood. Here we monitored calcium transients in zebrafish embryos as a proxy for ENE to assess the efficacy of transient pharmacological treatments able to either increase or decrease ENE. Increasing or decreasing ENE for 24 hour at 2 days post fertilization (dpf) promoted respectively an increase or decrease in the post-mitotic differentiation of the dopamine (DA) neurons in the telencephalon and in the olfactory bulb of zebrafish larvae at 6 dpf. This plasticity of dopaminergic specification occurs within a stable population of vMAT2 immuno-reactive cells, hence identifying an unanticipated biological marker for this pool of reserve cells, that can be recruited through ENE.

Modulating ENE also affected locomotion several days after the end of the treatments. In particular, the increase of ENE from 2 to 3 dpf promoted an hyperlocomotion in 6 dpf zebrafish larvae, reminiscent of endophenotypes reported for Attention Deficit with Hyperactivity Disorders and schizophrenia in zebrafish. These results provide a convenient framework to identify environmental factors that could disturb ENE as well as to study the molecular mechanisms linking ENE to the neurotransmitters specification, with clinical relevance for the pathogenesis of neurodevelopmental disorders.

Significance StatementO_LISpontaneous calcium spikes, used as a proxy for Embryonic Neuronal Excitability (ENE), are detected in the forebrain of embryonic zebrafish.
C_LIO_LITransients pharmacological treatments applied by balneation could be used to increase or decrease ENE.
C_LIO_LIThe post-mitotic differentiation of the dopaminergic phenotype is modulated by ENE in the zebrafish forebrain.
C_LIO_LIThe plasticity of the dopaminergic specification occurs within a reserve pool of vMAT2 immuno-reactive cells.
C_LIO_LITransient increase of ENE at the end of the embryonic period induces a hyperlocomotion, a phenotype associated with ADHD and schizophrenia in this model.
C_LIO_LIOur results open clinically relevant perspectives to study the pathogenesis of neurodevelopmental disorders in zebrafish.
C_LI Epitope-tagging of the endogenous murine BiP/GRP78/Hspa5 locus allows direct analysis of the BiP interactome and protein misfolding in vivo BiP/GRP78, encoded by the Hspa5 gene, is the major HSP70 family member in the endoplasmic reticulum (ER) lumen, and controls ER protein folding. The essential functions of BiP in facilitating proper protein folding are mainly mediated through its dynamic interaction with unfolded or misfolded client proteins, and by serving as a negative regulator of the Unfolded Protein Response. A mechanistic understanding of the dynamics of BiP interaction with its protein partners is essential to understand ER biology, and therefore, we have sought to develop a tractable model to study misfolded protein interaction with BiP. For this purpose, we have used homologous recombination to insert a 3xFLAG epitope tag into the endogenous murine Hspa5 gene, just upstream from the essential KDEL signal necessary for ER localization of BiP. Tagging BiP in this way did not alter Hspa5 expression under basal or ER-stress induced conditions in hepatocytes ex vivo or in fibroblasts. Furthermore, the tag did not alter the cellular localization of BiP or its functionality. All of these findings in primary tissue culture were also confirmed in vivo in livers of heterozygous mice harboring one WT and one FLAG-tagged Hspa5 allele. Hepatocyte-specific BiP-FLAG modification did not alter liver function or UPR signaling. Importantly, immunoprecipitation with anti-FLAG antibody completely pulled down FLAG-tagged BiP from lysates of BiP-FLAG expressing livers. Affinity purification-mass spectrometry (AP-MS) of BiP-FLAG protein complexes isolated from the BiP-FLAG-expressing livers of tunicamycin (TM)- and vehicle-treated mice revealed a marked increase in interaction of glycoproteins with BiP-FLAG in response to inhibition of N-glycosylation due to TM-treatment, validating utility of our BiP-Flag mice as a tool to identify ER misfolded proteins in vivo. Significantly, our AP-MS analysis also provided in vivo evidence demonstrating that BiP-FLAG binds to UPR transducers IRE1 and PERK under basal conditions but is released upon TM-treatment to activate UPR. We have also employed this mouse model to demonstrate that proinsulin in pancreatic {beta} cells misfolds and interacts with BiP-FLAG in healthy mice. In summary, we generated a novel model that can be used to investigate the BiP interactome in vivo under physiological and pathophysiological conditions in a cell type-specific manner. This tool provides, for the first time, an unbiased approach to identify unfolded and misfolded BiP-client proteins, and a new approach to study ER protein misfolding in a cell-type and temporal manner to uncover the role of BiP in many essential ER processes. Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9 CRISPR-mediated genome editing has become a powerful tool for genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve the HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3' end of the MKK2 MAP Kinase gene to generate a BleoR-MKK2 fusion protein. Based on exogenous BleoR expression, the HDR-mediated knock-in efficiency was up to [~]40-fold greater when using a XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared to no HDR enhancement or NHEJ silencing. Furthermore, this corporation of HDR enhancer overexpression and NHEJ repression also resulted in 7-fold fewer CRISPR-induced Insertions and Deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammalians for improving CRISPR-mediated gene knock-in efficiency. A vesicle microrheometer for high-throughput viscosity measurements of lipid and polymer membranes Viscosity is a key property of cell membranes that controls mobility of embedded proteins and membrane remodeling. Measuring it is challenging because existing approaches involve complex experimental designs and/or models, and the applicability of some is limited to specific systems and membrane compositions. As a result there is scarcity of data and the reported values for membrane viscosity vary by orders of magnitude for the same system. Here, we show how viscosity of bilayer membranes can be obtained from the transient deformation of giant unilamellar vesicles. The approach enables a non-invasive, probe-independent and high-throughput measurement of the viscosity of bilayers made of lipids or polymers with a wide range of compositions and phase state. Pure lipid and single-phase mixed bilayers are found to behave as Newtonian fluids with strain-rate independent viscosity, while phase-separated and diblock-copolymers systems exhibit shear-thinning in the explored range of strain rates 1-2000 s-1. The results also reveal that electrically polarized bilayers can be significantly more viscous than charge-neutral bilayers. These findings suggest that biomembrane viscosity is a dynamic property that can be actively modulated not only by composition but also by membrane polarization, e.g., as in action potentials. A TIR-1/SARM1 phase transition underlies p38 immune pathway activation in the C. elegans intestine Intracellular signaling regulators can be concentrated into membrane-free, higher-ordered protein assemblies to initiate protective responses during stress -- a process known as phase transition. Here, we show that a phase transition of the Caenorhabditis elegans Toll/interleukin-1 receptor domain protein (TIR-1), an NAD+ glycohydrolase homologous to mammalian sterile alpha and TIR motif-containing 1 (SARM1), underlies p38 PMK-1 immune pathway activation in C. elegans intestinal epithelial cells. Through visualization of fluorescently labeled TIR-1/SARM1 protein, we demonstrate for the first time that physiologic stresses, both pathogen and non-pathogen, induce multimerization of TIR-1/SARM1 into visible puncta within intestinal epithelial cells. In vitro enzyme kinetic analyses revealed that, like mammalian SARM1, the NAD+ glycohydrolase activity of C. elegans TIR-1 is dramatically potentiated by protein oligomerization and a phase transition. Accordingly, C. elegans with genetic mutations that specifically block either multimerization or the NAD+ glycohydrolase activity of TIR-1/SARM1 fail to induce p38 PMK phosphorylation, are unable to increase immune effector expression, and are dramatically susceptible to bacterial infection. Finally, we demonstrate that the TIR-1/SARM1 phase transition is modified by dietary cholesterol, revealing a new adaptive response that allows a metazoan host to anticipate pathogen threats during micronutrient deprivation, a time of relative susceptibility to infection. When cholesterol is limited, TIR-1/SARM1 oligomerizes into puncta in intestinal epithelial cells and engages its NAD+ glycohydrolase activity, which increases p38 PMK-1 phosphorylation, and primes immune effector induction in a manner that promotes pathogen clearance from the intestine during a subsequent infection. Thus, a phase transition of TIR-1/SARM1 as a prerequisite for its NAD+ glycohydrolase activity is strongly conserved across millions of years of evolution and is essential for diverse physiological processes in multiple cell types.