SUFI: An automated approach to spectral unmixing of fluorescent multiplex images captured in mouse and postmortem human brain tissues Multispectral fluorescence imaging coupled with linear unmixing is a form of image data collection and analysis that uses multiple fluorescent dyes - each measuring a specific biological signal - that are simultaneously measured and subsequently "unmixed" to provide a read-out for each individual signal. This strategy allows for measuring multiple signals in a single data capture session - for example, multiple proteins or RNAs in tissue slices or cultured cells, but can often result in mixed signals and bleed-through problems across dyes. Existing spectral unmixing algorithms are not optimized for challenging biological specimens such as postmortem human brain tissue, and often require manual intervention to extract spectral signatures. We therefore developed an intuitive, automated, and flexible package called SUFI: spectral unmixing of fluorescent images (https://github.com/LieberInstitute/SUFI). This package unmixes multispectral fluorescence images by automating the extraction of spectral signatures using Vertex Component Analysis, and then performs one of three unmixing algorithms derived from remote sensing. We demonstrate these remote sensing algorithms performance on four unique biological datasets and compare the results to unmixing results obtained using ZEN Black software (Zeiss). We lastly integrate our unmixing pipeline into the computational tool dotdotdot that is used to quantify individual RNA transcripts at single cell resolution in intact tissues and perform differential expression analysis of smFISH data, and thereby provide a one-stop solution for multispectral fluorescence image analysis and quantification. In summary, we provide a robust, automated pipeline to assist biologists with improved spectral unmixing of multispectral fluorescence images.
3CAC: improving the classification of phages and plasmids in metagenomic assemblies using assembly graphs MotivationBacteriophages and plasmids usually coexist with their host bacteria in microbial communities and play important roles in microbial evolution. Accurately identifying sequence contigs as phages, plasmids, and bacterial chromosomes in mixed metagenomic assemblies is critical for further unravelling their functions. Many classification tools have been developed for identifying either phages or plasmids in metagenomic assemblies. However, only two classifiers, PPR-Meta and viralVerify, were proposed to simultaneously identify phages and plasmids in mixed metagenomic assemblies. Due to the very high fraction of chromosome contigs in the assemblies, both tools achieve high precision in the classification of chromosomes but perform poorly in classifying phages and plasmids. Short contigs in these assemblies are often wrongly classified or classified as uncertain.

ResultsHere we present 3CAC, a new three-class classifier that improves the precision of phage and plasmid classification. 3CAC starts with an initial three-class classification generated by existing classifiers and improves the classification of short contigs and contigs with low confidence classification by using proximity in the assembly graph. Evaluation on simulated metagenomes and on real human gut microbiome samples showed that 3CAC outperformed PPR-Meta and viralVerify in both precision and recall, and increased F1-score by 10-60 percentage points.

AvailabilityThe 3CAC software is available on https://github.com/Shamir-Lab/3CAC.

Contactrshamir@tau.ac.il

Supplementary informationSupplementary data are available at Bioinformatics online.
Adolescent Alcohol Binge-Drinking induces delayed appearance of behavioral defects in mice Adolescence is a developmental period characterized by significant changes in brain architecture and behavior. The immaturity of the adolescent brain is associated with heightened vulnerability to exogenous agents, including alcohol. Alcohol is the most consumed drug among teenagers, and binge-drinking during adolescence is a major public health concern. Studies have suggested that adolescent alcohol exposure (AAE) may interfere with the maturation of frontal brain regions and lead to long-lasting behavioral consequences. In this study, we used a mouse model of AAE in which adolescent mice reach high blood alcohol concentration after voluntary binge-drinking. In order to assess short- and long-term consequences of AAE, a battery of behavioral tests was performed during late adolescence and during adulthood. We showed that AAE had no short-term effect on young mice behavior but rather increased anxiety- and depressive-like behaviors, as well as alcohol consumption during adulthood. Moreover, alcohol binge-drinking during adolescence dramatically decreased recognition memory performances and behavioral flexibility in both adult males and females. Furthermore, we showed that voluntary consumption of alcohol during adolescence did not trigger any major activation of the innate immune system in the prefrontal cortex (PFC). Together, our data suggest that voluntary alcohol binge-drinking in adolescent mice induces a delayed appearance of behavioral impairments in adulthood. Crystal structures of alphavirus nonstructural protein 4 (nsP4) reveal an intrinsically dynamic RNA-dependent RNA polymerase fold. Alphaviruses such as Ross River virus (RRV), chikungunya virus, Sindbis virus (SINV), and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 and 1.9 [A]. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogendeuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Key PointsO_LICrystal structures of alphavirus nsP4 RNA polymerase domain from RRV and SINV.
C_LIO_LInsP4 protein is highly dynamic with an intrinsically disordered N-terminal domain.
C_LIO_LIOptimized RNA elongation activity assay to facilitate antiviral discovery.
C_LI Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats implicated unique Lewis X epitopes in mammals The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans. Toxicity Profile of Artesunate in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel. Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics. The "LINC" in between Delta40p53-miRNA axis in the regulation of cellular processes Recently, we showed that {Delta}40p53, the translational isoform of p53, can inhibit cell growth independently of p53 by regulating microRNAs. Here, we explored the role of {Delta}40p53 in regulating the lncRNA-miRNA axis. Based on early research from our laboratory, we selected LINC00176 for further exploration. LINC00176 levels were affected by the overexpression and knockdown of {Delta}40p53. Further, under DNA damage, ER stress, and glucose deprivation, LINC00176 was upregulated in HCT116 p53-/- cells (harboring only {Delta}40p53) compared to HCT116 p53+/+ cells. Additionally, ChIP and RNA stability assays revealed that {Delta}40p53 transactivates LINC00176 transcriptionally and stabilizes it post-transcriptionally. We ectopically overexpressed and knocked down LINC00176 in HCT116 p53-/- cells, which affected proliferation, cell viability, and the expression of epithelial markers. Finally, RNA pulldown revealed that LINC00176 sequesters several putative miRNA targets. These results provide important insights into the pivotal role of {Delta}40p53 in regulating the lncRNA-miRNA axis.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/489891v2_ufig1.gif" ALT="Figure 1">
View larger version (31K):
org.highwire.dtl.DTLVardef@1035f85org.highwire.dtl.DTLVardef@74185borg.highwire.dtl.DTLVardef@1eb3704org.highwire.dtl.DTLVardef@168ebab_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Model depicting the regulation of {Delta}40p53-LINC00176-miRNA axis

(A) In absence of {Delta}40p53, LINC00176 levels do not increase which leads to increased pool of free miRNAs which probably binds to several mRNA targets preventing their function, ultimately resulting in increased cell proliferation. (B) However, when {Delta}40p53 is present it upregulates LINC00176 levels both at the level of transcription and post-transcription, which sequesters oncogenic miRNAs preventing it to bind to their respective mRNA targets, which ultimately results in decreased cell proliferation.

C_FIG High expression of VRT2 increases the number of rudimentary basal spikelets in wheat Spikelets are the fundamental building blocks of Poaceae inflorescences and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat, the central spikelets produce the most and largest grains, while spikelet size gradually decreases acro- and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient correlates with the developmental age of spikelets, however the basal spikelets are developed first and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterise gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central and basal sections of a single spike than between any section belonging to consecutive developmental timepoints. We found that SVP MADS-box transcription factors, including VRT-A2, are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-class SEP1 genes. Based on multi-year field trials and transgenic lines, we show that higher expression of VRT-A2 in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modelling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programmes results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain new insights into developmental genetics pathways of grass inflorescences.

One sentence summaryLarge transcriptional gradients exist within a wheat spike and are associated with rudimentary basal spikelet development, resulting in the characteristic lanceolate shape of wheat spikes. Egress of human cytomegalovirus through multivesicular bodies Human Cytomegalovirus (HCMV) can infect a variety of cell types by using virions of varying glycoprotein compositions. It is still unclear how this diversity is generated, but spatio-temporally separated envelopment and egress pathways might play a role. So far, one egress pathway has been described in which HCMV particles are individually enveloped into small vesicles and are subsequently exocytosed continuously. However, some studies have also found enveloped virus particles inside multivesicular structures but could not link them to productive egress or degradation pathways.

We used a novel 3D-CLEM workflow allowing us to investigate these structures in HCMV morphogenesis and egress at high spatio-temporal resolution. We found that multiple envelopment events occurred at individual vesicles leading to multiviral bodies (MViBs), which subsequently traversed the cytoplasm to release virions as intermittent bulk pulses at the plasma membrane to form extracellular virus accumulations (EVAs). Our data support the existence of a novel bona fide HCMV egress pathway, which opens the gate to evaluate divergent egress pathways in generating virion diversity. Species-specific developmental timing dictates expansion of the avian wing skeletal pattern A fundamental question in biology is how species-specific rates of embryonic development are controlled in differently sized species. To address this problem, we compared wing development in the quail and the larger chick. We reveal that pattern formation (humerus to digits) is faster in the quail wing as determined by the earlier activation of 5 Hox genes, termination of developmental organisers (Shh and Fgf8) and the laying down of the skeleton (Sox9). Using interspecies tissue grafts, we show that developmental timing can be reset during a critical window in which retinoic acid signalling is active. Accordingly, retinoic acid can switch developmental timing between the quail and chick, and also between the chick and the larger turkey. We show that the incremental growth rate is equivalent between all three species, suggesting that differences in the expansion of the skeletal pattern are governed primarily by the tempo of development. Our findings have implications for how development is timed both within and between different species. Temperature Increase Drives Critical Slowing Down of Fish Ecosystems Fish ecosystems perform ecological functions that are critically important for the sustainability of marine ecosystems, such as global food security and carbon stock. During the 21st century, significant global warming caused by climate change has created pressing challenges for fish ecosystems that threaten species existence and global ecosystem health. Here, we study a coastal fish community in Maizuru Bay, Japan, and investigate the relationships between fluctuations of ST, abundance-based species interactions and salient fish biodiversity. Observations show that a local 20% increase in temperature from 2002 to 2014 underpins a long-term reduction in fish diversity ([~]25%) played out by some native and invasive species (e.g. Chinese wrasse) becoming exceedingly abundant; this causes a large decay in commercially valuable species (e.g. Japanese anchovy) coupled to an increase in ecological productivity. The fish community is analyzed considering five temperature ranges to understand its atemporal seasonal sensitivity to ST changes, and long-term trends. An optimal information flow model is used to reconstruct species interaction networks that emerge as topologically different for distinct temperature ranges and species dynamics. Networks for low temperatures are more scale-free compared to ones for intermediate (15-20{degrees}C) temperatures in which the fish ecosystem experiences a first-order phase transition in interactions from locally stable to metastable and globally unstable for high temperatures states as suggested by abundance-spectrum transitions. The dynamic dominant eigenvalue of species interactions shows increasing instability for competitive species (spiking in summer due to intermediate-season critical transitions) leading to enhanced community variability and critical slowing down despite higher time-point resilience. Native competitive species whose abundance is distributed more exponentially have the highest total directed interactions and are keystone species (e.g. Wrasse and Horse mackerel) for the most salient links with cooperative decaying species. Competitive species, with higher eco-climatic memory and synchronization, are the most affected by temperature and play an important role in maintaining fish ecosystem stability via multitrophic cascades (via cooperative-competitive species imbalance), and as bioindicators of change. More climate-fitted species follow temperature increase causing larger divergence divergence between competitive and cooperative species. Decreasing dominant eigenvalues and lower relative network optimality for warmer oceans indicate fishery more attracted toward persistent oscillatory states, yet unpredictable, with lower cooperation, diversity and fish stock despite the increase in community abundance due to non-commercial and venomous species. We emphasize how changes in species interaction organization, primarily affected by temperature fluctuations, are the backbone of biodiversity dynamics and yet for functional diversity in contrast to taxonomic richness. Abundance and richness manifest gradual shifts while interactions show sudden shift. The work provides data-driven tools for analyzing and monitoring fish ecosystems under the pressure of global warming or other stressors. Abundance and interaction patterns derived by network-based analyses proved useful to assess ecosystem susceptibility and effective change, and formulate predictive dynamic information for science-based fishery policy aimed to maintain marine ecosystems stable and sustainable. Global Constraints within the Developmental Program of the Drosophila Wing Organismal development is a complex process, involving a vast number of molecular constituents interacting on multiple spatio-temporal scales in the formation of intricate body structures. Despite this complexity, development is remarkably reproducible and displays tolerance to both genetic and environmental perturbations. This robust-ness implies the existence of hidden simplicities in developmental programs. Here, using the Drosophila wing as a model system, we develop a new quantitative strategy that enables a robust description of biologically salient phenotypic variation. Analyzing natural phenotypic variation across a highly outbred population, and variation generated by weak perturbations in genetic and environmental conditions, we observe a highly constrained set of wing phenotypes. Remarkably, the phenotypic variants can be described by a single integrated mode that corresponds to a non-intuitive combination of structural variations across the wing. This work demonstrates the presence of constraints that funnel environmental inputs and genetic variation into phenotypes stretched along a single axis in morphological space. Our results provide quantitative insights into the nature of robustness in complex forms while yet accommodating the potential for evolutionary variations. Methodologically, we introduce a general strategy for finding such invariances in other developmental contexts. 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. Ineffectual AEC1 differentiation from KRT8hi transitional state without fibrosis is associated with fatal COVID-19 ARDS ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. Unifying Single-Channel Permeability from Rare-Event Sampling and Steady-State Flux Various all-atom molecular dynamics (MD) simulation methods have been developed to compute free energies and crossing rates of ions and small molecules through ion channels. However, a systemic comparison across different methods is scarce. Using a carbon nanotube as a model of small conductance ion channel, we computed the single-channel permeability for potassium ion using umbrella sampling, Markovian milestoning, and steady-state flux under applied voltage. We show that a slightly modified inhomogeneous solubility-diffusion equation yields a single-channel permeability consistent with the mean first passage time (MFPT)-based method. For milestoning, applying cylindrical and spherical bulk boundary conditions yield consistent MFPT if factoring in the effective bulk concentration. The sensitivity of the MFPT to the output frequency of collective variables is highlighted using the convergence and symmetricity of the inward and outward MFPT profiles. The consistent transport kinetic results from all three methods demonstrated the robustness of MD-based methods in computing ion channel permeation. The advantages and disadvantages of each technique are discussed, focusing on the future applications of milestoning in more complex systems. Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems. Classification of Pacemaker Dynamics in the Mouse Intestine by Field Potential Microimaging The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals. A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. Genetic architecture of natural variation of cardiac performance in flies Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP)1. Genome Wide Associations Studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict Transcription Factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans. Meningeal dendritic cells-astrocytes interactions elevate the kynurenine metabolic pathway to sustain neuropathic pain Neuropathic pain is triggered by injury to the somatosensory system, and is one of the most important types of chronic pain. Nevertheless, critical pathophysiological mechanisms that maintain neuropathic pain are poorly understood. Here, we show that neuropathic pain is abrogated when the kynurenine metabolic pathway (KYNPATH) initiated by the enzyme indoleamine 2,3-dioxygenase (IDO) is ablated pharmacologically or genetically. Mechanistically, it was found that IDO upregulation in dendritic cells that accumulate in the dorsal root leptomeninges led to increased levels of kynurenine (Kyn) in the spinal cord, where Kyn is metabolized by astrocytes-expressed kynurenine-3-monooxygenase into a pro-nociceptive metabolite 3-hydroxykynurenine. In conclusion, these data reveal a novel role for KYNPATH as an important factor maintaining neuropathic pain during neuroimmune-glia cells interactions. This novel paradigm offers potential new targets for drug development against this type of chronic pain. phenopype: A phenotyping pipeline for Python O_LIDigital images are an intuitive way to capture, store and analyze organismal phenotypes. Many biologists are taking images to collect high-dimensional phenotypic information from specimens, which are key to investigating complex ecological, evolutionary and developmental phenomena, such as relationships between trait diversity and ecosystem function, multivariate natural selection, or developmental plasticity. As a consequence, images are being collected at ever increasing rates, but extraction of the contained phenotypic information still poses a veritable analytical bottleneck.
C_LIO_LIphenopype is a high throughput phenotyping pipeline for the programming language Python that aims at alleviating this bottleneck. The package facilitates immediate extraction of high dimensional phenotypic data from laboratory grade digital images with low levels of background noise and complexity. At the core, phenopype provides functionality for rapid signal processing based image preprocessing and segmentation, followed by trait extraction, visualization and numerous options for data export. The functionality is provided by wrapping low-level Python computer vision libraries (e.g. OpenCV) into accessible functions, facilitating their incorporation into scientific image analysis workflows. In addition, phenopype provides a project management ecosystem to further simplify rapid data collection and to increase reproducibility.
C_LIO_LIphenopype offers two different workflows that support users during different stages of scientific image analysis. The low-throughput workflow uses regular Python syntax and has greater flexibility at the cost of reproducibility, which is suitable for prototyping during the initial stage of a research project. The high-throughput workflow allows users to specify and store image-specific settings for analysis in human-readable YAML format, and then execute all functions in one step by means of an interactive parser. This approach facilitates rapid program-user interactions during batch processing, and greatly increases scientific reproducibility.
C_LIO_LIOverall, phenopype intends to make the features of powerful but technically involved low-level CV libraries available to biologists with little or no Python coding experience. Therefore, phenopype is aiming to augment, rather than replace the utility of existing Python CV libraries, allowing biologists to focus on rapid and reproducible data collection. Furthermore, image annotations produced by phenopype can be used as training data, thus presenting a stepping stone towards the application of deep learning architectures.
C_LI Naming Human Diseases: Ethical Principles of Curating Exclusive Substitute for Inopportune Nosology BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Force redistribution during clathrin-mediated endocytosis revealed by new phase-separating force sensors Forces are central to countless cellular processes, yet in vivo force measurement at the molecular scale remains difficult if not impossible. During clathrin-mediated endocytosis, forces produced by the actin cytoskeleton are transmitted to the plasma membrane by a multi-protein coat for membrane deformation. However, the magnitudes of these forces remain unknown. Here, we present new in vivo force sensors that phase separate under force. We measured the forces on the fission yeast HIP1R homologue End4p, a protein that links the membrane to the actin cytoskeleton. End4p is under [~]15 pN force near the actin cytoskeleton, [~] 11 pN near the clathrin lattice, and [~]9 pN near the plasma membrane. Our results demonstrate that forces are collected and redistributed across the endocytic machinery.

One-Sentence SummaryPhase-separating coiled-coil force sensors reveal an unexpected mode of force transduction during endocytosis in vivo. Identification of differentially distributed gene expression and distinct sets of cancer-related genes identified by changes in mean and variability There is increasing evidence that changes in the variability or overall distribution of gene expression are important both in normal biology and in diseases, particularly cancer. Genes whose expression differs in variability or distribution without a difference in mean are ignored by traditional differential expression-based analyses. Using a Bayesian hierarchical model that provides tests for both differential variability and differential distribution for bulk RNA-seq data, we report here an investigation into differential variability and distribution in cancer. Analysis of eight paired tumour-normal datasets from The Cancer Genome Atlas confirms that differential variability and distribution are able to identify cancer-related genes. We further demonstrate that differential variability identifies cancer-related genes that are missed by differential expression analysis, and that differential expression and differential variability identify functionally distinct sets of genes. These results suggest that differential variability analysis may provide insights into genetic aspects of cancer that would not be revealed by differential expression, and that differential distribution analysis may allow for more comprehensive identification of cancer-related genes than analyses based on changes in mean or variability alone. Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2 The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form.