N-glycan chitobiose core biosynthesis by Agl24 strengthens the hypothesis of an archaeal origin of the eukaryal N-glycosylation Protein N-glycosylation is the most common posttranslational modifications found in all three domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in eukaryotes. Here, we report the identification of a thermostable archaeal beta-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed the function as an inverting {beta}-D-GlcNAc-(1[-&gt;]4)--D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed strong similarities between Agl24 and both the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which catalyze the identical or a similar process, respectively. Our data, complemented by phylogenetic analysis of Alg13 and Alg14, revealed similar sequences in Asgardarchaeota, further supporting the hypothesis that the Alg13/14 homologs in eukaryotes have been acquired during eukaryogenesis.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/427365v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@13d1ffdorg.highwire.dtl.DTLVardef@1ff48d0org.highwire.dtl.DTLVardef@1056e75org.highwire.dtl.DTLVardef@196ff58_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIFirst identification and characterization of a thermostable {beta}-D-GlcNAc-(1[-&gt;]4)--D-GlcNAc-diphosphodolichol glycosyltransferase (GT family 28) in Archaea.
C_LIO_LIA highly conserved histidine, within a GGH motif in Agl24, Alg14, and MurG, is essential for function of Agl24.
C_LIO_LIAgl24-like homologs are broadly distributed among Archaea.
C_LIO_LIThe eukaryotic Alg13 and Alg14 are closely related to the Asgard homologs, suggesting their acquisition during eukaryogenesis.
C_LI
Targeting conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, and especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wildtype viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. Here, we analyze in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses dont stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications.
Somewhere I belong: phylogenetic comparative methods and machine learning to investigate the evolution of a species-rich lineage of parasites AO_SCPLOWBSTRACTC_SCPLOWA substantial portion of biodiversity evolved through adaptive radiation. However, the effects of explosive speciation on species interactions remain poorly understood. Metazoan parasites infecting radiating host lineages could improve our knowledge because of their intimate host relationships. Yet limited molecular, phenotypic, and ecological data discourage multivariate analyses of evolutionary patterns and encourage the use of discrete characters. Here, we assemble new molecular, morphological, and host range data widely inferred from a species-rich lineage of parasites (Cichlidogyrus, Platyhelminthes: Monogenea) infecting cichlid fishes to address data scarcity. We infer a multi-marker (28S/18S rDNA, ITS1, COI mtDNA) phylogeny of 58/137 species and characterise major lineages through synapomorphies inferred from mapping morphological characters. We predict the phylogenetic position of species without DNA data through shared character states, a combined molecular-morphological phylogenetic analysis, and a classification analysis with support vector machines. Based on these predictions and a cluster analysis, we assess the systematic informativeness of continuous characters, search for continuous equivalents for discrete characters, and suggest new characters for morphological traits not analysed to date. We also model the attachment/reproductive organ and host range evolution using the data of 136/137 described species and multivariate phylogenetic comparative methods (PCMs). We show that discrete characters can mask phylogenetic signals but can be key for characterising species groups. Regarding the attachment organ morphology, a divergent evolutionary regime for at least one lineage was detected and a limited morphological variation indicates host and environmental parameters affecting its evolution. However, moderate success in predicting phylogenetic positions, and a low systematic informativeness and high multicollinearity of morphological characters call for a revaluation of characters included in species characterisations. 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. Structural basis for targeting the human T-cell leukemia virus Tax oncoprotein and syntenin-1 interaction using a small molecule Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Although ATL is a well-characterized T-cell neoplasm, linked to intermittent expression of the viral Tax-1 protein, there is currently no strategy to target Tax-1 functions using small molecules. Here, we report a comprehensive interaction map between Tax-1 and human PDZ domain-containing proteins (hPDZome). We show that Tax-1 interacts with more than one-third of hPDZome components, including proteins involved in cell cycle, cell-cell junctions, cytoskeleton organization, and membrane complex assembly. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the structural basis of the interaction between the C-terminal PDZ binding motif (PBM) of Tax-1, and the PDZ domains of syntenin-1, an evolutionary conserved hub that controls exosome trafficking. Finally, we have used confocal imaging, molecular modelling, NMR and mammalian cell-based assays to demonstrate that the Tax-1/syntenin-1 interaction is amenable to small-molecule inhibition. Altogether, our study highlights the biological significance of Tax-PDZ interactome and its interplay with exosome formation. It shows a direct link between extracellular vesicles and HTLV-1 transmission, providing a novel framework for the design of targeted therapies for HTLV-1-induced diseases. Mouse lemur, a new primate model to investigate cardiac pacemaker activity. BackgroundGray mouse lemurs (Microcebus murinus) are one of the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since Microcebus murinus replicates similar senescence processes of humans, it constitutes a useful model also for studying cardiovascular dysfunctions. However, its cardiac physiology is unknown. Thus, we investigated sinoatrial node (SAN) pacemaker activity of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.

Methods and ResultsWe recorded the heart automaticity in freely moving mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of electrical impulses in the SAN tissue. Conversely, their ventricular depolarization is more similar to large mammals. Mouse lemur SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (ICa,L) and T-type (ICa,T) calcium currents. Moreover, mouse lemur SAN myocytes display spontaneous ryanodine receptor (RyR)-dependent Ca2+ transients. Using muscle-derived stem cells (MDSCs) from the Microcebus murinus skeletal muscle, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).

ConclusionsIn conclusion, our characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans, and for pharmacologic testing of molecules that could assist in modulating age-related dysfunctions of automaticity. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. A positively tuned voltage indicator reveals electrical correlates of calcium activity in the brain Neuronal activity is routinely recorded in vivo using genetically encoded calcium indicators (GECIs) and 2-photon microscopy, but calcium imaging is poorly sensitive for single voltage spikes under typical population imaging conditions, lacks temporal precision, and does not report subthreshold voltage changes. Genetically encoded voltage indicators (GEVIs) offer better temporal resolution and subthreshold sensitivity, but 2-photon detection of single spikes in vivo using GEVIs has required specialized imaging equipment. Here, we report ASAP4b and ASAP4e, two GEVIs that brighten in response to membrane depolarization, inverting the fluorescence-voltage relationship of previous ASAP-family GEVIs. ASAP4b and ASAP4e feature 180% and 210% fluorescence increases to 100-mV depolarizations, respectively, as well as modestly prolonged deactivation and high photostability. We demonstrate single-trial detection of spikes and oscillations in vivo with standard 1 and 2-photon imaging systems, and confirm improved temporal resolution in comparison to calcium imaging on the same equipment. Thus, ASAP4b and ASAP4e GEVIs extend the uses of existing imaging equipment to include multiunit voltage imaging in vivo.

One Sentence SummaryPositively tuned ASAP voltage indicators facilitate imaging of electrical activity in the brain. Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land. Comparative toxicogenomics of glyphosate and Roundup herbicides by mammalian stem cell-based genotoxicity assays and molecular profiling in Sprague-Dawley rats Whether glyphosate-based herbicides (GBHs) are more potent than glyphosate alone at activating cellular mechanisms, which drive carcinogenesis remains controversial. As GBHs are more cytotoxic that glyphosate, we reasoned they may also be more capable of activating carcinogenic pathways. We tested this hypothesis by comparing the effects of glyphosate with Roundup GBHs both in vitro and in vivo. First, glyphosate was compared with representative GBHs namely MON 52276 (EU), MON 76473 (UK) and MON 76207 (USA) using the mammalian stem cell-based ToxTracker system. Here, MON 52276 and MON 76473, but not glyphosate and MON 76207, activated oxidative stress and unfolded protein responses. Second, molecular profiling of liver was performed in female Sprague-Dawley rats exposed to glyphosate or MON 52276 (both at 0.5, 50, 175 mg/kg bw/day glyphosate) for 90 days. MON 52276 but not glyphosate increased hepatic steatosis and necrosis. MON 52276 and glyphosate altered the expression of genes in liver reflecting TP53 activation by DNA damage and circadian rhythm regulation. Genes most affected in liver were similarly altered in kidneys. Small RNA profiling in liver showed decreased amounts of miR-22 and miR-17 from MON 52276 ingestion. Glyphosate decreased mir-30 while miR-10 levels were increased. DNA methylation profiling of liver revealed 5,727 and 4,496 differentially methylated CpG sites between the control and glyphosate and MON 52276 exposed animals respectively. Apurinic/apyrimidinic DNA damage formation in liver was increased with glyphosate exposure. Altogether, our results show that Roundup formulations cause more biological changes linked with carcinogenesis than glyphosate. Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germ line specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESCs self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification. TeamTree analysis: a new approach to evaluate scientific production Advances in science and technology depend on the work of research teams and the publication of results through peer-reviewed articles representing a growing socio-economic resource. Current methods to mine the scientific literature regarding a field of interest focus on content, but the workforce credited by authorship remains largely unexplored, and appropriate measures of scientific production are debated. Here, a new bibliometric approach named TeamTree analysis is introduced that visualizes the development and composition of the workforce driving a field. A new citation-independent measure that scales with the H index estimates impact based on publication record, genealogical ties and collaborative connections. This author-centered approach complements existing tools to mine the scientific literature and to evaluate research across disciplines. Mathematical Modeling and Analysis of Mitochondrial Retrograde Signaling Dynamics Mitochondria, semi-autonomous eukaryotic organelles, participate in energy production and metabolism, making mitochondrial quality control crucial. As most mitochondrial proteins are encoded by nuclear genes, quality control depends on proper mitochondria-nucleus communication, designated mitochondrial retrograde signaling. Early studies focused on retrograde signaling participants and specific gene knockouts. However, mitochondrial signal modulation remains elusive. Using yeast, we simulated signal propagation following mitochondrial damage and proposed a mathematical model based ordinary differential equations. Mitochondrial retrograde signaling decisions were described using a Boolean model. Dynamics were analyzed through an ordinary differential equation-based model and extended to evaluate the model response to noisy damage signals. Simulation revealed localized protein concentration dynamics, including waveforms, frequency response, and robustness under noise. Retrograde signaling is bistable with three localized steady states, and increased damage compromises robustness. We elucidated mitochondrial retrograde signaling, thus providing a basis for drug design against yeast and fungi.

Author SummaryThe yeast RTG pathway regulates mitochondrial metabolism and mitochondrial quality through passing mitochondrial signal to the nucleus to modulate gene expressions. Using microscopic data of RTG proteins translocation from cytosol to nucleus, the parameters were found by fitting 16 knockout conditions simulated from the proposed differential equation-based model. Further, through dose response, ultrasensitivity, frequency response and noise interference, we demonstrated a switch-like property of RTG pathway activation, a capacity charging property of RTG protein translocation, a low pass filter property of signals in different frequencies, and noise amplification due to mitochondrial signal. The proposed mathematical model enables us to understand the dynamics and mechanisms in mitochondrial retrograde signaling in yeast and provides potential antifungal treatment strategies. Penalized Logistic Regression Analysis for Genetic Association Studies of Binary Phenotypes IntroductionIncreasingly, logistic regression methods for genetic association studies of binary phenotypes must be able to accommodate data sparsity, which arises from unbalanced case-control ratios and/or rare genetic variants. Sparseness leads to maximum likelihood estimators (MLEs) of log-OR parameters that are biased away from their null value of zero and tests with inflated type 1 errors. Different penalized-likelihood methods have been developed to mitigate sparse-data bias. We study penalized logistic regression using a class of log-F priors indexed by a shrinkage parameter m to shrink the biased MLE towards zero.

MethodsWe propose a two-step approach to the analysis of a genetic association study: first, a set of variants that show evidence of association with the trait is used to estimate m; and second, the estimated m is used for log-F -penalized logistic regression analyses of all variants using data augmentation with standard software. Our estimate of m is the maximizer of a marginal likelihood obtained by integrating the latent log-ORs out of the joint distribution of the parameters and observed data. We consider two approximate approaches to maximizing the marginal likelihood: (i) a Monte Carlo EM algorithm (MCEM) and (ii) a Laplace approximation (LA) to each integral, followed by derivative-free optimization of the approximation.

ResultsWe evaluate the statistical properties of our proposed two-step method and compared its performance to other shrinkage methods by a simulation study. Our simulation studies suggest that the proposed log-F -penalized approach has lower bias and mean squared error than other methods considered. We also illustrate the approach on data from a study of genetic associations with "super senior" cases and middle aged controls.

Discussion/ConclusionWe have proposed a method for single rare variant analysis with binary phenotypes by logistic regression penalized by log-F priors. Our method has the advantage of being easily extended to correct for confounding due to population structure and genetic relatedness through a data augmentation approach. The interplay of chromatin phase separation and lamina interactions in nuclear organization The genetic material of eukaryotes is segregated into transcriptionally active euchromatin and silent heterochromatin compartments. The spatial arrangement of chromatin compartments evolves over the course of cellular life in a process that remains poorly understood. The latest nuclear imaging experiments reveal a number of dynamical signatures of chromatin which are reminiscent of active multi-phase liquids. This includes the observations of viscoelastic response, coherent motions, Ostwald ripening and coalescence of chromatin compartments. There is also growing evidence that liquid-liquid phase separation of protein and nucleic acid components is the underlying mechanism for the dynamical behavior of chromatin. In order to dissect the organizational and dynamical implications of chromatins liquid behavior, we have devised a phenomenological field-theoretic model of nucleus as a multi-phase condensate of liquid chromatin types. Employing the liquid chromatin model of Drosophila nucleus, we have carried out an extensive set of simulations with an objective to shed light on the dynamics and chromatin patterning observed in the latest nuclear imaging experiments. Our simulations reveal the emergence of experimentally detected mesoscale chromatin channels and spheroidal droplets which arise from the dynamic interplay of chromatin type to type interactions and intermingling of chromosomal territories. We also quantitatively reproduce coherent motions of chromatin domains observed in displacement correlation spectroscopy measurements which are explained within the framework of our model by phase separation of chromatin types operating within constrained intra-chromosomal and inter-chromosomal boundaries. Finally, we illuminate the role of heterochromatin-lamina interactions in the nuclear organization by showing that these interactions enhance the mobility of euchromatin and indirectly introduce correlated motions of heterochromatin droplets.

SIGNIFICANCE STATEMENTThe latest super-resolution imaging experiments have revealed a surprisingly dynamic and stochastic nature of chromatin in eukaryotic nuclei which is reminiscent of multi-phase fluid behavior. As a result, the concepts from the theory of complex fluids such as phase separation, viscoelasticity, and droplet nucleation have found widespread utility in understanding salient features of nuclear organization. In order to understand and disentangle the complex interplay of forces that contribute to the emergent patterns of organization and dynamics, we have devised a phenomenological field-theoretic model of nucleus as a multi-phase condensate of liquid chromatin types. Armed with a mesoscopic model of nuclear chromatin, we have shed light on the distinct dynamical and structural contributions of chromatin type interactions intermingling of chromosomal territories and lamina binding. We also shed light on the dynamical heterogeneity and coherent motions of chromatin domains which are fully captured by an interplay of micro-phase separation of chromatin types and lamina binding. RNA polymerase II is recruited to DNA double-strand breaks for dilncRNA transcription in Drosophila DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Designing viral diagnostics with model-based optimization Diagnostics, particularly for rapidly evolving viruses, stand to benefit from a principled, measurement-driven design that harnesses machine learning and vast genomic data--yet the capability for such design has not been previously built. Here, we develop and extensively validate an approach to designing viral diagnostics that applies a learned model within a combinatorial optimization framework. Concentrating on CRISPR-based diagnostics, we screen a library of 19,209 diagnostic-target pairs and train a deep neural network that predicts, from RNA sequence alone, diagnostic signal better than contemporary techniques. Our model then makes it possible to design assays that are maximally sensitive over the spectrum of a viruss genomic variation. We introduce ADAPT (https://adapt.guide), a system for fully-automated design, and use ADAPT to design optimal diagnostics for the 1,933 vertebrate-infecting viral species within 2 hours for most species and 24 hours for all but 3. We experimentally show ADAPTs designs are sensitive and specific down to the lineage level, including against viruses that pose challenges involving genomic variation and specificity. ADAPTs designs exhibit significantly higher fluorescence and permit lower limits of detection, across a viruss entire variation, than the outputs of standard design techniques. Our model-based optimization strategy has applications broadly to viral nucleic acid diagnostics and other sequence-based technologies, and, paired with clinical validation, could enable a critically-needed, proactive resource of assays for surveilling and responding to pathogens. The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging. ASURAT: functional annotation-driven unsupervised clustering of single-cell transcriptomes MotivationSingle-cell RNA sequencing (scRNA-seq) analysis reveals heterogeneity and dynamic cell transitions. However, conventional gene-based analyses require intensive manual curation to interpret the biological implications of computational results. Hence, a theory for efficiently annotating individual cells is necessary.

ResultsWe present ASURAT, a computational pipeline for simultaneously performing unsupervised clustering and functional annotation of disease, cell type, biological process, and signaling pathway activity for single-cell transcriptomic data, using correlation graph-based decomposition of genes based on database-derived functional terms. We validated the usability and clustering performance of ASURAT using scRNA-seq datasets for human peripheral blood mononuclear cells, which required fewer manual curations than existing methods. Moreover, we applied ASURAT to scRNA-seq and spatial transcriptome datasets for small cell lung cancer and pancreatic ductal adenocarcinoma, identifying previously overlooked subpopulations and differentially expressed genes. ASURAT is a powerful tool for dissecting cell subpopulations and improving biological interpretability of complex and noisy transcriptomic data.

AvailabilityA GPLv3-licensed implementation of ASURAT is on GitHub (https://github.com/keita-iida/ASURAT). Impedance scaling for gold and platinum microelectrodes ObjectiveElectrical measurement of the activity of individual neurons is a primary goal for many invasive neural electrodes. Making these "single unit" measurements requires that we fabricate electrodes small enough so that only a few neurons contribute to the signal, but not so small that the impedance of the electrode creates overwhelming noise or signal attenuation. Thus, neuroelectrode design often must strike a balance between electrode size and electrode impedance, where the impedance is often assumed to scale linearly with electrode area.

Approach and main resultsHere we study how impedance scales with neural electrode area and find that the 1 kHz impedance of Pt electrodes (but not Au electrodes) transitions from scaling with area (r-2) to scaling with perimeter (r-1) when the electrode radius falls below 10 microns. This effect can be explained by the transition from planar to spherical diffusion behavior previously reported for electrochemical microelectrodes. Significance: These results provide important intuition for designing small, single unit recording electrodes. Specifically, for materials where the impedance is dominated by a pseudo-capacitance that is associated with a diffusion limited process, the total impedance will scale with perimeter rather than area when the electrode size becomes comparable with the diffusion layer thickness. For Pt electrodes this transition occurs around 10 um radius electrodes. At even lower frequencies (1 Hz) impedance approaches a constant. This transition to r-1 scaling implies that electrodes with a pseudo-capacitance can be made smaller than one might expect before thermal noise or voltage division limits the ability to acquire high-quality single-unit recordings. The response network of HSP70 defines vulnerabilities in cancer cells with the inhibited proteasome Human neoplasias are often addicted to the proteasome machinery. However, cancers have evolved efficient response mechanisms to overcome proteasome inhibition with bortezomib and carfilzomib - drugs approved for multiple myeloma treatment. To understand these responses we investigated proteome changes upon the proteasome inhibition with carfilzomib - in multiple myeloma, normal fibroblasts, and cancers of lung, colon, and pancreas. A pathway-oriented siRNA screen based on the proteomics results showed that molecular chaperones, autophagy- and endocytosis-related proteins are cancer-specific vulnerabilities combined with carfilzomib. Targeting of HSPA1A/B (HSP70 family chaperones) most specifically sensitized cancer cells and patient-derived organoids to the proteasome inhibition. A high level of HSPA1A/B mRNA correlated with a low proteasome activity in cancer patient tissues and is a risk factor in cancer patients with a low proteasome expression. Mechanistically, HSPA1A/B governed autophagy, unfolded protein response, endocytic trafficking, and chaperoned the proteasome machinery, suppressing the effect of the proteasome inhibition, but did not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, downregulation of NRF1 most specifically decreased the viability of cancer cells with the inhibited proteasome and HSP70. Propelling and perturbing appendages together facilitate strenuous ground self-righting Terrestrial animals must self-right when overturned on the ground, but this locomotor task is strenuous. To do so, the discoid cockroach often pushes its wings against the ground to begin a somersault which rarely succeeds. As it repeatedly attempts this, the animal probabilistically rolls to the side to self- right. During winged self-righting, the animal flails its legs vigorously. Here, we studied whether wing opening and leg flailing together facilitate strenuous ground self-righting. Adding mass to increase hind leg flailing kinetic energy increased the animals self-righting probability. We then developed a robot with similar strenuous self-righting behavior and used it as a physical model for systematic experiments. The robots self-righting probability increased with wing opening and leg flailing amplitudes. A potential energy landscape model revealed that, although wing opening did not generate sufficient kinetic energy to overcome the high pitch potential energy barrier to somersault, it reduced the barrier for rolling, facilitating the small kinetic energy from leg flailing to probabilistically overcome it to self-right. The model also revealed that the stereotyped body motion during self-righting emerged from physical interaction of the body and appendages with the ground. Our work demonstrated the usefulness of potential energy landscape for modeling self-righting transitions.

Impact statementAnimals can, and robots should, use different types of appendages together to propel and perturb themselves to self-right when overturned, a strenuous yet crucial locomotor task. Repeated introduction of micropollutants enhances microbial succession despite stable degradation patterns The increasing-volume release of micropollutants into natural surface waters has raised great concern due to their environmental accumulation. Persisting micropollutants can impact multiple generations of organisms, but their microbially-mediated degradation and their influence on community assembly remain understudied. Here, freshwater microbes were treated with several common micropollutants, alone or in combination, and then transferred every 5 days to fresh medium containing the same micropollutants to mimic the repeated exposure of microbes. Metabarcoding of 16S rRNA gene makers was chosen to study the succession of bacterial assemblages following micropollutant exposure. The removal rates of micropollutants were then measured to assess degradation capacity of the associated communities. The degradation of micropollutants did not accelerate over time but altered the microbial community composition. Community assembly was dominated by stochastic processes during early exposure, via random community changes and emergence of seedbanks, and deterministic processes later in the exposure, via advanced community succession. Early exposure stages were characterized by the presence of sensitive microorganisms such as Actinobacteria and Planctomycetes, which were then replaced by more tolerant bacteria such as Bacteroidetes and Gammaproteobacteria. Our findings have important implication for ecological feedback between microbe-micropollutants under anthropogenic climate change scenarios. β-glucan imprinting remodels macrophage function in response to environmental cues In vitro, exposure of human primary monocytes to the fungal {beta}-glucan enhances their pro-inflammatory responsiveness towards several pathogens. Yet, the role of environmental condition of this process remains unclear. Here we found that {beta}-glucan-induced innate immune memory counteract the anti-inflammatory status of the macrophages. In response to {beta}-glucan imprinting, M-CSF-(M2-like-) macrophages increase their pro-inflammatory responsiveness to secondary stimuli associated with decrease of the M-CSF differentiation hallmarks. In contrast, in GM-CSF-(M1-like-) environment, {beta}-glucan imprinting reduced the pro-inflammatory canonical feature of the macrophages, together with their terminal differentiation marks. Comparing M-CSF and GM-CSF environment, we observed that {beta}-glucan-imprinted macrophages present comparable functions in terms of cytokine responses, phagocytosis, oxidative burst, and angiogenesis. This effect is mediated through Dectin-1 and associated with altered expression of the master regulators of macrophage terminal differentiation, IRF5 and IRF3. {beta}-glucan-induced innate immune memory skews the commitment of the macrophages in complex environment towards similar and less terminally differentiated cells. Together, these observations suggest a potential therapeutic role for {beta}-glucan-induced modulation of innate memory in different pathological contexts. In vitro pectate lyase activity and carbon uptake assays and whole genome sequencing of Bacillus amyloliquefaciens subsp. plantarum strains for a pectin defective pathway The pectin lyase activity of 59 Bacillus amyloliquefaciens subsp. plantarum (Bap) strains was tested in vitro on Pectate Agar (PA) and Tris-Spizizen Salts (TSS) medium. Bap strains were cultured on TSA medium and washed three times with sterile water before the inoculation on PA media. Higher and lower pectate lyase activity were observed in six (AP193, AP203, AP299, AP80, AP102, and AP52) and four (AP 194, AP214, AP215, and AP305) Bap strains compared to other Bap strains. A total of 12 Bap strains (AP67, AP71, AP77, AP78, AP85, AP102, AP108, AP135, AP143, AP189, AP192, and AP193) grew vigorously on TSS medium. A total of six Bap strains (AP194, AP204, AP214, AP216, AP219, and HD73) had lower growth compared to other Bap strains. Pectin (1%) were used for in vitro PA and TSS medium. Pectate lyase and utilization activity were not found in Bacillus thuringiensis subsp. kurstaki strain HD73 compared to Bap strains. A draft genome sequence for strains AP194 and AP214 that were negative for pectin utilization were generated using an Illumina MiSeq. RAST analysis revealed that the pectin-associated gene altronate hydrolase (uxaA) absent in AP 214 strain. Multiple amino acid alignments of exuT and uxuB gene sequence showed dissimilarities among AP194, AP214, and reference Bap strains.