Snrpb, the CCMS gene, is required in neural crest cells for proper splicing of genes essential for craniofacial morphogenesis Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). We show that Snrpb heterozygous embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5 splice site strength. We found increased exon-skipping in negative regulators of the P53-pathway, increased levels of nuclear P53, P53-target genes. However, removing TrP53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations.
Transcriptomic analysis of temporal shifts in berry development between two grapevine cultivars of the Pinot family reveals potential genes controlling ripening time BackgroundGrapevine cultivars of the Pinot family represent clonally propagated mutants with major phenotypic and physiological differences, such as different colour or shifted ripening time, as well as changes in important viticultural traits. Specifically, the cultivars  Pinot Noir (PN) and  Pinot Noir Precoce (PNP, early ripening) flower at the same time, but vary in the beginning of berry ripening (veraison) and, consequently, harvest time. In addition to genotype, seasonal climatic conditions (i.e. high temperatures) also affect ripening times. To reveal possible regulatory genes that affect the timing of veraison onset, we investigated differences in gene expression profiles between PN and PNP throughout berry development with a closely meshed time series and over two separate years.

ResultsThe difference in the duration of berry formation between PN and PNP was quantified to be approximately two weeks under the growth conditions applied, using plant material with a proven PN and PNP clonal relationship. Clusters of co-expressed genes and differentially expressed genes (DEGs) were detected which reflect the shift in the timing of veraison onset. Functional annotation of these DEGs fit to observed phenotypic and physiological changes during berry development. In total, we observed 3,342 DEGs in 2014 and 2,745 DEGs in 2017 between PN and PNP, with 1,923 DEGs across both years. Among these, 388 DEGs were identified as veraison-specific and 12 were considered as berry ripening time regulatory candidates. The expression profiles revealed two candidate genes for ripening time control which we designated VviRTIC1 and VviRTIC2 (VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP.

ConclusionsMany of the 1,923 DEGs show highly similar expression profiles in both cultivars if the patterns are aligned according to developmental stage. In our work, putative genes differentially expressed between PNP and PN which could control ripening time as well as veraison-specific genes were identified. We point out connections of these genes to molecular events during berry development and discuss potential candidate genes which may control ripening time. Two of these candidates were observed to be differentially expressed in the early berry development phase. Several down-regulated genes during berry ripening are annotated as auxin response factors / ARFs. Conceivably, general changes in auxin signaling may cause the earlier ripening phenotype of PNP.
Fine-scale adaptations to environmental variation and growth strategies drive phyllosphere Methylobacterium diversity. Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium was structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20 {degrees}C or 30 {degrees}C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100km; between forests) and small geographical scales (<1.2km within forests), among host tree species, and was dynamic over seasons. By measuring growth of 79 isolates at different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities, in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This works paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus.

ImportanceMethylobacterium is a bacterial group tied to plants. Despite its ubiquity and importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial scales (within 1.2 km). Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains in lab conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasted growth strategies in response to environmental variations. 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. Perceptual gating of a brainstem reflex facilitates speech understanding in human listeners Navigating "cocktail party" situations by enhancing foreground sounds over irrelevant background information is typically considered from a cortico-centric perspective. However, subcortical circuits, such as the medial olivocochlear (MOC) reflex that modulates inner ear activity itself, have ample opportunity to extract salient features from the auditory scene prior to any cortical processing. To understand the contribution of auditory subcortical nuclei and the cochlea, physiological recordings were made along the auditory pathway while listeners differentiated non(sense)-words and words. Both naturally-spoken and intrinsically-noisy, vocoded speech -- filtering that mimics processing by a cochlear implant--significantly activated the MOC reflex, whereas listening to speech-in-background noise revealed instead engagement of midbrain and cortical resources. An auditory periphery model reproduced these speech degradation-specific effects, providing a rationale for goal-directed gating of the MOC reflex to enhance representation of speech features in the auditory nerve. Our data reveals the co-existence of two strategies in the auditory system that may facilitate speech understanding in situations where the speech signal is either intrinsically degraded or masked by extrinsic auditory information. Opposing mechanisms of competition determine species invasions and functional diversity Understanding how species phenotypic differences affect competition is key to explaining community assembly and predicting biodiversity responses. Many studies overlook the variable effects that species trait differences can have on the direction of competitive exclusion, which reverses depending on the specific mechanism at play. We performed a comprehensive trait-based study of an ant invasion integrating morphological, dietary, physiological and behavioral analyses. We found that trait differences between invasive and resident species were not only associated with niche differences which promoted the coexistence of dissimilar species, but also competitive ability differences which acted in the opposite fashion. Furthermore, competition along separate trait axes led to complex and contrasting patterns in the invaded assemblages, where species were at once similar (clustered) in some traits but also dissimilar (overdispersed) in others. Our results reveal that different aspects of phenotype may distinctly modulate the effect of competition in structuring ecological communities and functional diversity. Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.

MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.

ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.

ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors. Ecological interactions in breast cancer: Cell facilitation promotes growth and survival under drug pressure The interplay of positive and negative interactions between drug sensitive and resistant cells determines the efficacy of treatment in heterogeneous cancer cell populations. We developed isogenic estrogen receptor positive (ER+) breast cancer cell lineages sensitive and resistant to ribociclib-induced CDK4/6 inhibition to investigate cell interactions under selective pressure. During treatment with ribociclib alone or in combination with endocrine therapies, sensitive cells survive and proliferate better when grown together with resistant cells than when grown in monoculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays detected estrone, a less active metabolite, in medium taken from each lineage but detected highly active estradiol only from resistant cells. Because sensitive cells are more dependent on estrogen signaling than resistant cells, they benefit from the increase in local estradiol from resistant cells, leading to facilitated growth when grown under treatment in coculture. We developed a suite of mathematical models to quantify the strength of competition and facilitation between cell lines and evaluate the effects of treatments that modify these interactions. These analyses support the conclusion that resistant cell lineages facilitate growth of sensitive lineages during therapy through the increased production and sharing of local estradiol and themselves suffer from increased competition and suppression as a result. These models show that blocking facilitation has the potential to control both resistant and sensitive cell populations and inhibit the emergence of a refractory population. This combination of experiments and models shows that interactions within heterogeneous cancer cell populations can combine positive and negative interactions to shape the dynamics of resistance. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. OncoLoop: A network-based precision cancer medicine framework At present, prioritizing cancer treatments at the individual patient level remains challenging, and performing co-clinical studies using patient-derived models in real-time is often not feasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework to predict and validate drug sensitivity in human tumors and their pre-existing high-fidelity (cognate) model(s) by leveraging perturbational profiles of clinically-relevant oncology drugs. As proof-of-concept, we applied OncoLoop to prostate cancer (PCa) using a series of genetically-engineered mouse models (GEMMs) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of published cohorts using Master Regulator (MR) conservation analysis revealed that most patients were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated, including in two cognate allografts and one cognate patient-derived xenograft (PDX). OncoLoop is a highly generalizable framework that can be extended to other cancers and potentially other diseases.

Significance StatementOncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround co-clinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. Systematic temporal analysis of peripheral blood transcriptomes using TrendCatcher identifies early and persistent neutrophil activation as a hallmark of severe COVID-19 Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there is a lack of methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic datasets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal datasets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk RNA-seq and scRNA-seq time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways as well as impaired type I interferon (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS- CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets. Comparative proteome signatures of trace samples by multiplexed Data-Independent Acquisition Single cell transcriptomics has revolutionized our understanding of basic biology and disease. Since transcript levels often do not correlate with protein expression, it is crucial to complement transcriptomics approaches with proteome analyses at single cell resolution. Despite continuous technological improvements in sensitivity, mass spectrometry-based single cell proteomics ultimately faces the challenge of reproducibly comparing the protein expression profiles of thousands of individual cells. Here, we combine two hitherto opposing analytical strategies, DIA and Tandem-Mass-Tag (TMT)-multiplexing, to generate highly reproducible, quantitative proteome signatures from ultra-low input samples. While conventional, data-dependent shotgun proteomics (DDA) of ultra-low input samples critically suffers from the accumulation of missing values with increasing sample-cohort size, data-independent acquisition (DIA) strategies do usually not take full advantage of isotope-encoded sample multiplexing. We developed a novel, identification-independent proteomics data-analysis pipeline that allows to quantitatively compare DIA-TMT proteome signatures across hundreds of samples independent of their biological origin, and to identify cell types and single protein knockouts. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. These data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultra-low input samples, such as single cells. 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. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI Multiparametric profiling of cellular lipid metabolism in hypercholesterolemia Systematic insight into cellular dysfunctions can improve understanding of disease etiology, risk assessment and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validated this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrated lipid storage data into a novel readout, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlated positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explained hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking from a few mls of blood and is applicable to dissect metabolic disorders, such as hypercholesterolemia.

MotivationWe have limited information on how cellular lipid uptake and processing differ between individuals and influence the development of metabolic diseases, such as hypercholesterolemia. Available assays are labor intensive, require skilled personnel and are difficult to scale to higher throughput, making it challenging to obtain systematic functional cell-based data from individuals. To overcome this problem, we established a scalable automated analysis pipeline enabling reliable quantification of multiple cellular readouts, including lipid uptake, storage and mobilization, from different white blood cell populations. This approach provides new personalized insights into the cellular basis of hypercholesterolemia and obesity.

Graphical Abstract

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org.highwire.dtl.DTLVardef@9222c1org.highwire.dtl.DTLVardef@27e289org.highwire.dtl.DTLVardef@89ba82org.highwire.dtl.DTLVardef@33c9cd_HPS_FORMAT_FIGEXP  M_FIG C_FIG Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades. Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants. An integrative approach leads to the discovery of a novel anti-leukemic peptide from human milk Chemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets are relatively resistant to standard chemotherapy. It is therefore important to identify new agents with sensitivity and selectivity towards leukemic cells, while having less systemic toxicity. Peptide-based therapeutics has gained much attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide having 3+charges and alpha-helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no effect on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane to enter the cytoplasm and cause leakage of lactate dehydrogenase, thus acting in a membranolytic manner. Importantly, HMP-S7 exhibited anti-leukemic effect against patient-derived leukemic cells ex vivo. In conclusion, HMP-S7 is a selective anti-leukemic peptide with promise which requires further validation in preclinical and clinical studies.

TeaserIn silico screening of naturally occurring human milk peptides discovers a new anticancer peptide that kills leukemic cells in vitro and ex vivo. Outcome-selective reinstatement is predominantly context-independent, and associated with c-Fos activation in the posterior dorsomedial striatum Research from human and animal studies has found that responding that has been successfully reduced following treatment can return upon exposure to certain contexts. An individual in recovery from alcohol use disorder, for example, might relapse to drinking upon visiting their favourite bar. However, most of these data have been derived from experiments involving a single (active) response, and the context-dependence of returned responding in situations involving choice between multiple actions and outcomes is less well-understood. We thus investigated how outcome-selective reinstatement - a procedure involving choice between multiple actions - was affected by altering the physical context in rats. In Experiment 1, rats were trained over 6 days to press a left lever for one food outcome (pellets or sucrose) and a right lever for the other outcome. Then, rats received an extinction session in either the same context (A) as lever press training, or in a different context (B). Rats were tested immediately (5 minutes) after extinction in Context A or B such that there were four groups in total: AAA, ABB, ABA, and AAB. Reinstatement testing consisted of one food outcome being delivered  freely (i.e. unearned by lever pressing and unsignalled by cues) to the food magazine every 4 minutes in the following order: Sucrose, Pellet, Pellet, Sucrose. Selective reinstatement was considered intact if pellet delivery increased pressing selectively on the pellet lever, and sucrose delivery selectively increased pressing on the sucrose lever. This result (Reinstated > Nonreinstated) was observed for rats in group AAA and ABB, but not rats in groups ABA and AAB. Experiment 2 was conducted identically, except that rats received two extinction sessions over two days and tested one day later. This time, all groups demonstrated intact outcome-selective reinstatement regardless of context. Analysis of c-Fos expression in several brain regions revealed that only c-Fos expression in the posterior dorsomedial striatum (pDMS) was related to intact reinstatement performance. Overall, these results suggest that outcome-selective reinstatement is predominantly context-independent, and that intact reinstatement is related to neuronal activity in the pDMS.

HighlightsO_LIOutcome-selective reinstatement is predominantly context-independent
C_LIO_LIOutcome-selective reinstatement is entirely context-independent after multiple extinction sessions
C_LIO_LIOutcome-selective reinstatement increases c-Fos expression in dorsomedial striatum
C_LIO_LIc-Fos expression in orbitofrontal cortex and dorsal hippocampus is unaffected by selective reinstatement.
C_LI An hidden Markov model to estimate homozygous-by-descent probabilities associated with nested layers of ancestors Inbreeding results from the mating of related individuals and has negative consequences because it brings together deleterious variants in one individual. Genomic estimates of the inbreeding coefficients are preferred to pedigree-based estimators as they measure the realized inbreeding levels and they are more robust to pedigree errors. Several methods identifying homozygous-by-descent (HBD) segments with hidden Markov models (HMM) have been recently developed and are particularly valuable when the information is degraded or heterogeneous (e.g., low-fold sequencing, low marker density, heterogeneous genotype quality or variable marker spacing). We previously developed a multiple HBD class HMM where HBD segments are classified in different groups based on their length (e.g., recent versus old HBD segments) but we recently observed that for high inbreeding levels with many HBD segments, the estimated contributions might be biased towards more recent classes (i.e., associated with large HBD segments) although the overall estimated level of inbreeding remained unbiased. We herein propose a new model in which the HBD classification is modeled in successive nested levels with decreasing expected HBD segment lengths, the underlying exponential rates being directly related to the number of generations to the common ancestor. The non-HBD classes are now modeled as a mixture of HBD segments from later generations and shorter non-HBD segments (i.e., both with higher rates). The new model has improved statistical properties and performs better on simulated data compared to our previous version. We also show that the parameters of the model are easier to interpret and that the model is more robust to the choice of the number of classes. Overall, the new model results in an improved partitioning of inbreeding in different HBD classes and should be preferred. Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. Lifespan prolonging mechanisms and insulin upregulation without fat accumulation in long-lived reproductives of a higher termite Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it. Nucleosome-directed replication origin licensing independent of a consensus DNA sequence The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c.. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with new analysis of genome-wide ORC binding, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes. Population bottlenecks constrain host microbiome diversity and genetic variation impeding fitness It is becoming increasingly clear that microbial symbionts influence key aspects of their hosts fitness, and vice versa. This may fundamentally change our thinking about how microbes and hosts interact in influencing fitness and adaptation to changing environments. Here we explore how reductions in population size commonly experienced by threatened species influence microbiome diversity. Consequences of such reductions are normally interpreted in terms of a loss of genetic variation, increased inbreeding and associated inbreeding depression. However, fitness effects of population bottlenecks might also be mediated through microbiome diversity, such as through loss of functionally important microbes. Here we utilise 50 Drosophila melanogaster lines with different histories of population bottlenecks to explore these questions. The lines were phenotyped for egg-to-adult viability and their genomes sequenced to estimate genetic variation. The bacterial 16S rRNA gene was amplified in these lines to investigate microbial diversity. We found that 1) host population bottlenecks constrained microbiome richness and diversity, 2) core microbiomes of hosts with low genetic variation were constituted from subsets of microbiomes found in flies with higher genetic variation, 3) both microbiome diversity and host genetic variation contributed to host population fitness, 4) connectivity and robustness of bacterial networks was low in the inbred lines regardless of host genetic variation, 5) reduced microbial diversity was associated with weaker evolutionary responses of hosts in stressful environments, and 6) these effects were unrelated to Wolbachia density. These findings suggest that population bottlenecks reduce hologenomic variation (combined host and microbial genetic variation). Thus, while the current biodiversity crisis focuses on population sizes and genetic variation of eukaryotes, an additional focal point should be the microbial diversity carried by the eukaryotes, which in turn may influence host fitness and adaptability with consequences for the persistence of populations.

Author summaryIt is becoming increasingly clear that organisms and the microbes that live on or in them - their microbiome - affect each other in profound ways that we are just beginning to understand. For instance, a diverse microbiome can help maintain metabolic functions or fight pathogens causing diseases. A disrupted microbiome may be especially critical for animals and plants that occur in low numbers because of threats from e.g. human exploitation or climate change, as they may already suffer from genetic challenges such as inbreeding and reduced evolutionary potential. The importance of such a reduction in population size, called a bottleneck, on the microbial diversity and the potential interactive effects on host health remains unexplored. Here we experimentally test these associations by investigating the microbiomes of 50 inbred or non-inbred populations of vinegar flies. We found that restricting the population size constrain the hosts genetic variation and simultaneously decreases the diversity of the microbiome that they harbor, and that both effects were detrimental to host fitness. The microbial communities in inbred host populations were less robust than in their non-inbred counterparts, suggesting that we should increasingly consider the microbiome diversity, which may ultimately influence the health and persistence of threatened species. IRGM/Irgm1 Aggravates Progression of Atherosclerosis by Inducing Macrophage Apoptosis through the MAPK Signaling Pathway AimsAtherosclerosis underlies most cardiovascular diseases, among which acute coronary syndrome (ACS) caused by plaque rupture (PR) often leads to death. Immune-related GTPases (IRGM/Irgm1) have been extensively studied in inflammatory diseases, but their role in atherosclerosis is unclear. Determining how IRGM/Irgm1 promotes atherosclerotic plaque vulnerability will provide information for new biomarkers and/or therapeutic targets.

Methods and resultsWe identified ruptured and unruptured plaques by optical coherence tomography, and found that serum IRGM was highly expressed in patients with ST-segment elevation myocardial infarction in PR. We used ApoE-/-Irgm1+/+, ApoE-/-Irgm1+/- mice and chimeric mice to establish a model of advanced atherosclerosis. The results of pathological experiments showed that Irgm1 caused plaque necrosis. The ratio of neutral lipids and cholesterol crystals increases, while the content of collagen fibers decreases, aggravating the destabilization of atherosclerotic plaques. In vitro, we used multiple approaches to confirm that Irgm1 promotes macrophage apoptosis by promoting the production of reactive oxygen species and activating the MAPK signaling pathway.

ConclusionsIRGM may be a potential risk factor for PR. Mechanistic studies have shown that IRGM/Irgm1 contributes to the formation and rupture of fragile plaques. This is partly mediated by the induction of macrophage apoptosis via the MAPK signaling pathway. IRGM may offer new strategies for early treatment of ACS.

Translation viewOur findings indicate that IRGM/Irgm1 contributes to formation and rupture of vulnerable plaques. It suggests that IRGM may provide a new target for the early treatment of ACS. Muscle Activation Profile During Perturbed Walking is Modulated According to Body State During an unexpected loss of balance, avoiding a fall requires people to readjust their footing rapidly and effectively. We investigated the activation patterns of the ankle and knee muscles, and muscle fiber type recruitment resulting from unannounced, medio-lateral (i.e., right/left) horizontal-surface walking perturbations in twenty healthy adults (27.00{+/-}2.79 years, 10 females). Surface electromyography (sEMG) total spectral power for specific frequency bands (40-60Hz, 60-150Hz, 150-250Hz, 250-400Hz and 400-1000Hz), from tibialis anterior (TA) and vastus lateralis (VL) muscles were analyzed. Compared to non-perturbed walking, we found a significant increase in the total spectral power of lower-extremity muscles during the first 3 seconds after perturbation. When two feet were on the ground in time of perturbation we found a different muscle fiber type recruitment pattern between VL and TA muscles. This was not significant for perturbations implemented when one foot was on the ground. Our findings suggest that muscle operating frequency is modulated in real time to fit body state and somatosensory input in the context of functional goal requirements such as a rapid change of footing in response to unexpected loss of balance in single and double-support phases of gait.

New & NoteworthyTo study muscle spectral profiles in response to loss of balance, we investigated the dynamics of muscle spectral power changes, across different frequency bands after unannounced mechanical perturbations during walking. We showed increased activation of high-frequency motor units of the lower-limb muscles, subside 3 seconds after perturbation. Differences in power increase of specific frequency bands suggest that muscle activation is modulated in real time to fit body state in the context of functional goal requirements.