DeCalciOn: A hardware system for real-time decoding of in-vivo calcium imaging data Epifluorescence miniature microscopes ("miniscopes") are widely used for in vivo calcium imaging of neural population activity. Imaging data is usually collected while subjects are engaged in a task and stored for later offline analysis, but emerging techniques for online imaging offer potential for novel real-time experiments in which closed-loop interventions (such as neurostimulation or sensory feedback) are triggered at short latencies in response to neural population activity. Here we introduce DeCalciOn, a plug-and-play hardware device for online population decoding of in vivo calcium signals that can trigger closed-loop feedback at millisecond latencies, and is compatible with miniscopes that use the UCLA Data Acquisition (DAQ) interface. In performance tests, the position of rats (n=13) on a linear track was decoded in real time from hippocampal CA1 population activity by 24 linear classifiers. DeCalciOn required <2.5 ms after each end-of-frame to decode up to 1,024 calcium traces and trigger TTL control outputs. Decoding was most efficient using a  contour-free method of extracting traces from ROIs that were unaligned with neurons in the image, but  contour-based extraction from neuronal ROIs is also supported. DeCalciOn is an easy-to-use system for real-time decoding of calcium fluorescence that enables closed-loop feedback experiments in behaving animals.
Finger representations in primary somatosensory cortex are modulated during vibrotactile working memory task It is well-established that several cortical areas represent vibrotactile stimuli in somatotopic maps. However, whether such somatotopic representations remain active during the delay period of working memory (WM) tasks, i.e. in the absence of any tactile stimulation, is unknown. In our experiment, participants had to compare two tactile stimuli with different vibration frequencies that were separated by a delay period (memory condition) or they were exposed to identical stimuli but did not have to solve a WM task (no memory condition). Importantly, both vibrotactile stimuli were either applied to the right index or little finger. Analyzing the delay period, we identified a well-known fronto-parietal network of brain regions involved in WM but we did not find WM specific activity in S1. However, using multi-voxel pattern analysis (MVPA) and representational similarity analysis (RSA), we found that S1 finger representations were more dissimilar during the delay period of the WM condition than during the control condition. These results indicate that WM processes modulate the representational geometry of S1 suggesting that some aspects of the tactile WM content are represented in a somatotopic fashion.

HIGHLIGHTSO_LIMultivariate approaches were used to identify finger specific representational changes during vibrotactile frequency discrimination.
C_LIO_LIVibrotactile working memory modulates somatotopic finger representations in contralateral S1 during the delay period, i.e. in the absence of any tactile stimuli
C_LI
Titers of antibodies the receptor-binding domain (RBD) of ancestral SARS-CoV-2 are predictive for levels of neutralizing antibodies to multiple variants Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring. Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses. 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. 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. Extracellular Vesicle (EV) Mitochondria and EV/HSP27 Mixtures Protect the Ischemic Brain Endothelium Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of endothelial cell-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous heat shock protein 27 (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, which subsequently colocalized with the mitochondrial network of the recipient primary human BECs. BECs treated with m/lEVs increased relative ATP levels and displayed superior mitochondrial function. m/lEVs isolated from rotenone (mitochondrial complex I inhibitor) exposed BECs (RTN-m/lEVs) did not increase BECs ATP levels compared to naive m/lEVs. In contrast, RTN-sEVs functionality was minimally affected compared to naive sEVs. Compared to EV/HSP27 mixtures, PEG-DET/EV/HSP27 ternary mixtures increased HSP27 protein loading. (PEG-DET/HSP27)/EV ternary and EV/HSP27 binary mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

Highlights[tpltrtarr] Medium-to-large extracellular vesicles (m/lEVs), not small EVs contain mitochondria
[tpltrtarr]m/lEVs increased ATP and mitochondrial function in brain endothelial cells (BECs)
[tpltrtarr]Donor m/lEV mitochondria colocalized with host mitochondria in recipient BECs
[tpltrtarr]m/lEVs from rotenone-exposed BECs did not increase recipient BEC ATP levels
[tpltrtarr]PEG-DET/EV/HSP27 ternary mixtures increase exogenous HSP27 protein loading


Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/466491v4_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@155f618org.highwire.dtl.DTLVardef@8fa778org.highwire.dtl.DTLVardef@13bd44aorg.highwire.dtl.DTLVardef@ee771e_HPS_FORMAT_FIGEXP  M_FIG C_FIG Extending Bayesian Elo-rating to quantify the steepness of dominance hierarchies The steepness of dominance hierarchies provides information about the degree of competition within animal social groups and is thus an important concept in socioecology. The currently most widely-used metrics to quantify steepness are based on Davids scores (DS) derived from dominance interaction networks. One serious drawback of these DS-based metrics is that they are biased, i.e., network density systematically decreases steepness values. Here, we provide a novel approach to estimate steepness based on Elo-ratings, implemented in a Bayesian framework (STEER: Steepness estimation with Elo-rating). Our new metric has two key advantages. First, STEER is unbiased, precise and more robust to data density than DS-based steepness. Second, it provides explicit probability distributions of the estimated steepness coefficient, which allows uncertainty assessment. In addition, it relies on the same underlying concept and is on the same scale as the original measure, and thus allows comparison to existing published results. We evaluate and validate performance of STEER by means of experimentation on empirical and artificial data sets and compare its performance to that of several other steepness estimators. Our results suggest that STEER provides a considerable improvement over existing methods to estimate dominance hierarchy steepness. We provide an R package EloSteepness to calculate the new steepness measure, and also show an example of using steepness in a comparative analysis. 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. Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen While mutation is often deleterious, it can also be adaptive. Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study we investigate two factors that are predicted to influence the trade-off between the costs and benefits of mutation: ecology and genome size. To investigate the relationship between these factors and mutation rate we conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact ecology can have on the adaptive potential of bacterial pathogens. Structural insights into p300 regulation and acetylation-dependent genome organisation Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease. Herbivore life histories are altered by drought stress in their hosts plants Drought associated with climate change can stress plants, altering their interactions with phytophagous arthropods. Dryness not only impacts cultivated plants but also their parasites, which in some cases are favoured by drought. Herbivorous arthropods feeding on drought-stressed plants typically produce bigger offspring and develop faster. However, how much responses to abiotic factors differ among populations of a species remains poorly documented. Here, we evaluate variability among populations of a major agricultural pest, the two spotted spider mite, Tetranychus urticae, in response to drought-stress. We compare key life-history parameters of twelve populations that originate from climates ranging from wet and cool Atlantic locations to medium to dry hot Mediterranean locations. We evaluated how plant drought stress affects four life history traits: development time, fecundity, sex-ratio and emigration rate in an experiment comparing well-watered and drought-stressed bean plants. Mites feeding on drought-stressed plants developed faster and attempted to leave leaves less often, and young females were more fecund. The mites from wet temperate climates exhibited greater plasticity between the two water regimes than mites originating from dryer and hot climates. Thus, climate in the area of origin influences mite response to drought. Satellitome comparison of two oedipodine grasshoppers highlights the contingent nature of satellite DNA evolution BackgroundThe full catalogue of satellite DNA (satDNA) within a same genome constitutes the satellitome. The Library Hypothesis predicts that satDNA in relative species reflects that in their common ancestor, but the evolutionary mechanisms and pathways of satDNA evolution have never been analyzed for full satellitomes. We compare here the satellitomes of two Oedipodine grasshoppers (Locusta migratoria and Oedaleus decorus) which shared their most recent common ancestor about 22.8 Ma ago.

ResultsWe found that about one-third of their satDNA families (near 60 in every species) showed sequence homology and were grouped into 12 orthologous superfamilies. The turnover rate of consensus sequences was extremely variable among the 20 orthologous family pairs analyzed in both species. The satDNAs shared by both species showed poor association with sequence signatures and motives frequently argued as functional, except for short inverted repeats allowing short dyad symmetries and non-B DNA conformations. Orthologous satDNAs frequently showed different FISH patterns at both intra- and interspecific levels. We defined indices of homogenization and degeneration and quantified the level of incomplete library sorting between species.

ConclusionsOur analyses revealed that satDNA degenerates through point mutation and homogenizes through partial turnovers caused by massive tandem duplications (the so-called satDNA amplification). Remarkably, satDNA amplification increases homogenization, at intragenomic level, and diversification between species, thus constituting the basis for concerted evolution. We suggest a model of satDNA evolution by means of recursive cycles of amplification and degeneration, leading to mostly contingent evolutionary pathways where concerted evolution emerges promptly after lineages split. Global membrane tension is independent of polyacrylamide substrate stiffness Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked, and they are under tension. In-plane membrane tension and cortical tension both influence many cellular processes, including cell migration, division, and endocytosis. However, while actomyosin tension is regulated by substrate stiffness, how membrane tension responds to mechanical substrate properties is currently poorly understood. Here, we probed the effective membrane tension of neurons and fibroblasts cultured on glass and polyacrylamide substrates of varying stiffness using optical tweezers. In contrast to actomyosin-based traction forces, both peak forces and steady state tether forces of cells cultured on hydrogels were independent of substrate stiffness and did not change after blocking myosin II activity using blebbistatin, indicating that tether and traction forces are not directly linked. Peak forces in fibroblasts on hydrogels were about twice as high as those in neurons, indicating stronger membrane-cortex adhesion in fibroblasts. Steady state tether forces were generally higher in cells cultured on hydrogels than on glass, which we explain by a mechanical model. Our results provide new insights into the complex regulation of effective membrane tension and pave the way for a deeper understanding of the biological processes it instructs. Early postnatal activation of the hypoxia pathway disrupts β-cell function Hypoxic insults in the perinatal period can lead to persistent hyperinsulinism and profound hypoglycemia in newborns. We studied the impact of the hypoxia-inducible factor 1A (HIF1A) pathway on postnatal {beta}-cell function. Rat pups were treated daily between postnatal day (P)7 to P10 with adaptaquin (AQ), an inhibitor of prolyl hydroxylases, which stabilizes HIF1A. AQ-treated pups were hypoglycemic and had higher plasma insulin concentrations. Their islets had a decreased glucose threshold for insulin secretion, indicative of a delay in {beta}-cell postnatal functional maturation. Histology analyses revealed that AQ-treated pups had increased pancreatic insulin-positive area but no changes in the number of islets or number of {beta}-cells per islet, suggesting larger average {beta}-cell size. AQ-treated rat pups had decreased expression of cell cycle genes and decreased numbers of proliferating {beta}-cells. In conclusion, pharmacologic activation of the HIF1A pathway in the early postnatal period leads to hyperinsulinism, due to the persistence of a low glucose threshold for insulin secretion, and to decreased early postnatal {beta}-cell proliferation, suggesting it can impact adult {beta}-cell mass and diabetes risk. Tackling polypharmacology of kinase inhibitors and selecting chemical kinase probes by transcription factor activity profiling. Protein kinase inhibitors (PKI) evolved as promising drugs for multiple diseases. However, PKIs promiscuity causes polypharmacological effects challenging therapeutic development. We show that the polypharmacology of PKIs can be inferred from their impact on transcription factors (TF) lying at the apexes of signaling pathways. Using a high-content reporter assay, we evaluated TF activity profiles (TFAP) in cells treated with Akt, CDK, Aurora, Raf, MEK, and ERK inhibitors. For each kinase family, we found kinase-specific consensus TFAP signatures signifying the on-target PKI activity. Remarkably, proximal kinases had the highest similarity of their PKIs consensus signatures. Furthermore, we show that individual PKIs exhibited the consensus, "on-target signatures" within certain "specificity windows" and distinct "off-target signatures" at other concentrations. We also show that the TFAP signatures permit pinpointing the off-target PKI activity at irrelevant kinases and non-kinase effectors. Therefore, the TFAP approach provides clear-cut quantitative metrics for assessing PKI activity and concentration ranges where the on-target activity dominates cell response. By illuminating PKI biology invisible to target-based techniques, this effect-based approach streamlines the selection of kinase chemical probes and PKI drug prioritization. ZIKV disrupts placental ultrastructure and drug transporter expression in mice Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome. Long-term conservation effects of protected areas in stochastic population dynamics Terrestrial and marine protected areas are essential tools in mitigating anthropogenic impacts and promoting population persistence and resource sustainability. Adequately implemented protected areas (PAs) promote long-term conservation effects. Stochasticity causes fluctuations in the conservation effects of PAs, and so it is important to investigate the variabilities of these conservation effects to inform their long-term conservation effects. To investigate long-term conservation effects, I develop and analyze new models of stochastic processes that encompass the fluctuations generated by demographic or environmental stochasticity in PAs management. The stochastic model is built upon individual processes. In the model, density-independent mortality, migration between PAs and non-PAs, and site preferences characterize the features of the PA. The effect of PAs size is also examined. The long-term conservation effects are quantified using the coefficient of variation (CV) of population size in PAs, where a lower CV indicates higher robustness in stochastic variations. Typically, the results from this study demonstrate that sufficiently reduced density-independent mortality in PAs and high site preference and immigration rate of PA are likely to decrease the CV. However, different types of stochasticity induce rather different consequences: under demographic stochasticity, the CV is always reduced when PAs increase the population size therein, but an increased population size by PAs does not always decrease the CV under environmental stochasticity. The deterministic dynamics of the model are investigated, facilitating effective management decisions. CD155 and EndoA1 mediate growth and tissue invasion downstream of MAP4K4 in medulloblastoma cells The composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the FEME effector Endophilin A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells.

Graphical abstractc-MET activation upon HGF stimulation induces c-MET internalization and induces downstream MAP4K4 activity. (1) MAP4K4 is required downstream of activated c-MET for the maintenance of surface presentation of CD155 in activated cells. CD155 expression is required for MB cell migration, invasion and proliferation in the tissue context. (2) MAP4K4 is required downstream of activated c-MET to maintain membrane depolarization, possibly by regulating the surface localization of several ion channels and transporters. (3) MAP4K4 is required downstream of activated c-MET cause PM-proximal localization of FEME effector CIP4, FBP17 and CIN85. The FEME effector endophilin A is necessary for MB cell migration, invasion and dissemination. Effects of Ancestry and Hypopigmentation Alleles on Skin Color in a Caribbean Native American Population Interest in the genetic basis of variation in skin pigmentation in Native American populations led us to seek indigenous populations of the Western Hemisphere with African and minimal European admixture to study the effect of Native American ancestry on skin color. Admixture analysis from DNA collected from 458 individuals in the Kalinago territory of the Commonwealth of Dominica showed shared ancestry with East Asians at K=3 and 55% Native American, 32% African, and 11% European ancestry at K=6, the highest Native American ancestry of Caribbean populations. Skin pigmentation was 20 to 80 melanin units, averaging 46. Three albino individuals were homozygous for multi-nucleotide polymorphism OCA2NW273KV of African origin, whose population allele frequency was 0.03 and single allele effect size was -8 melanin units. Hypopigmenting allele frequencies for SLC24A5A111T and SLC45A2L374F were 0.14 and 0.05, whose single allele effect sizes were -6 and -3, respectively. Skin color plots of individuals lacking known hypopigmenting alleles suggests that Native American Ancestry reduced pigmentation by more than 20 melanin units (low and high estimates 21.8 and 28.5). Shared ancestry with East Asians at K=3 suggests potential sharing of one or more pigmentation alleles. The addition of FAIMS Increases Targeted Proteomics Sensitivity from FFPE Tumor Biopsies Mass spectrometry-based targeted proteomics allows objective protein quantitation of clinical biomarkers from a single section of formalin-fixed, paraffin-embedded (FFPE) tumor tissue biopsies. We combined high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) to increase assay sensitivity. The modular nature of the FAIMS source allowed direct comparison of the performance of FAIMS-PRM to PRM. Limits of quantitation were determined by spiking synthetic peptides into a human spleen matrix. In addition, 20 clinical samples were analyzed using FAIMS-PRM and the quantitation of HER2 was compared with that obtained with the Ventana immunohistochemistry assay. FAIMS-PRM improved the overall signal-to-noise ratio over that from PRM and increased assay sensitivity in FFPE tissue analysis for four (HER2, EGFR, cMET, and KRAS) of five proteins of clinical interest. FAIMS-PRM enabled sensitive quantitation of basal HER2 expression in breast cancer samples classified as HER2 negative by immunohistochemistry. Furthermore, we determined the degree of FAIMS-dependent background reduction and showed that this correlated with an improved lower limit of quantitation with FAIMS. FAIMS-PRM is anticipated to benefit clinical trials in which multiple biomarker questions must be addressed and the availability of tumor biopsy samples is limited. Nascent alt-protein chemoproteomics reveals a repressor of ribosome biogenesis Many unannotated microproteins and alternative proteins (alt-proteins) have recently been found to be co-encoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing active or stress-induced synthesis could play important cellular roles, here, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated unannotated alt-proteins, one of which is upregulated after DNA damage stress. We further defined MINAS-60 (MIcroprotein that Negatively regulates ASsembly of the pre-60S ribosomal subunit), a nucleolar localized alt-protein co-encoded with human RBM10.Depletion of MINAS-60 increases the amount of the mature 60S ribosomal subunit, consequently upregulating global protein synthesis and cell proliferation by repressing late-stage pre-60S assembly and export of the 60S ribosome subunit to the cytoplasm. Together, these results implicate MINAS-60 as a repressor of ribosome biogenesis, and demonstrate that chemoproteomics can enable generation of functional hypotheses for uncharacterized alt-proteins. 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. SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists The imaging of plant seedlings, fungal mycelia and bacterial colonies grown on Petri plates is common in phenotyping assays, and is typically done manually despite the procedures being time-consuming and laborious. The main reason for this is the still limited availability of existing automated phenotyping tools and facilities. Additionally, constructing a custom-made automated solution is a daunting task for most research groups specializing in biology.

Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment. SPIRO was designed for biologists by biologists; thus, its assembly does not require experience in engineering or programming and its operation is sufficiently intuitive to be carried out without training. SPIRO has a small footprint optimal for fitting into standard incubators for plants and microbes, and is equipped with an LED light source for imaging in the dark, thus allowing acquisition of photos under optimal growth conditions. SPIROs web-based user interface allows setting up experiments and downloading data remotely, without interfering with samples growth. SPIROs excellent image quality is optimal for automated image processing, which we demonstrate with two semi-automated assays for analysis of commonly used phenotypic traits: seed germination and root growth.

Moreover, the robot can be easily customized for a specific use, as all information about SPIRO, including the models for 3D-printed structural components, control software, and scripts for image analysis, are released under permissive open-source licenses. Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. Relative contributions of sex hormones, sex chromosomes, and gonads to sex differences in tissue gene regulation Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the Four Core Genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect and, lastly, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation, and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions.