Mouse frontal cortex mediates additive multisensory decisions To interpret the world and make accurate perceptual decisions, the brain must combine information across sensory modalities. For instance, it must combine vision and hearing to localize objects based on their image and sound. Probability theory suggests that evidence from multiple independent cues should be combined additively, and several studies support this hypothesis1-14. However, other studies suggest that mammals, particularly mice, can exhibit sensory bias during audiovisual integration15-19; furthermore, the cortical substrates of multisensory integration remain uncertain20. Here we show that to localize a stimulus, mice combine auditory and visual spatial cues additively, a computation supported by additive multisensory integration in frontal cortex. We developed an audiovisual localization task where mice turn a wheel to indicate the joint position of an image and a sound. Scanning optogenetic inactivation21-24 during stimulus presentation showed that visual cortex contributes unisensory information whereas frontal cortex contributes multisensory information to the mouses decision. Neuropixels recordings of >10,000 neurons indicated that neural activity in frontal area MOs (secondary motor cortex) reflects an additive combination of visual and auditory signals. An accumulator model2,25,26 applied to the sensory representations of MOs neurons reproduced both the observed choices and reaction times. Sensory signals in MOs were specific to trained mice, and indeed the same accumulator applied to naive mice failed to perform the task. These results suggest that frontal cortex adapts through learning to integrate information across sensory cortices, providing a signal that is then transformed into a binary decision by a downstream accumulator.
Control of Ca2+ signals by astrocyte nanoscale morphology at tripartite synapses Much of the Ca2+ activity in astrocytes is spatially restricted to microdomains and occurs in fine processes that form a complex anatomical meshwork, the so-called spongiform domain. A growing body of literature indicates that those astrocytic Ca2+ signals can influence the activity of neuronal synapses and thus tune the flow of information through neuronal circuits. Because of technical difficulties in accessing the small spatial scale involved, the role of astrocyte morphology on Ca2+ microdomain activity remains poorly understood. Here, we use computational tools and idealized 3D geometries of fine processes based on recent super-resolution microscopy data to investigate the mechanistic link between astrocytic nanoscale morphology and local Ca2+ activity. Simulations demonstrate that the nano-morphology of astrocytic processes powerfully shapes the spatio-temporal properties of Ca2+ signals and promotes local Ca2+ activity. The model predicts that this effect is attenuated upon astrocytic swelling, hallmark of brain diseases, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neurotransmitter release events, the model predicts that swelling hinders astrocytic signal propagation. Overall, this study highlights the influence of the complex morphology of astrocytes at the nanoscale and its remodeling in pathological conditions on neuron-astrocyte communication at so-called tripartite synapses, where astrocytic processes come into close contact with pre- and postsynaptic structures.

Table of ContentsO_ST_ABSMain PointsC_ST_ABSO_LIAstrocyte nano-morphology favors the compartmentalization of biochemical signals
C_LIO_LIThis compartmentalization promotes local Ca2+ activity and signal propagation robustness
C_LIO_LIIn contrast, its pathological remodeling upon swelling attenuates Ca2+ activity
C_LI

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O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/432635v4_fig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@22b80forg.highwire.dtl.DTLVardef@1621218org.highwire.dtl.DTLVardef@191e971org.highwire.dtl.DTLVardef@751637_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1:C_FLOATNO Proposed mechanisms that regulate astrocytic Ca2+ activity in perisynaptic astrocytic processes.

Our simulation results demonstrate that the nano-morphology of astrocytic processes, consisting in the alternation of nodes and shafts, favors the compartmentalization of biochemical signals. This compartmentalization promotes local Ca2+ activity and signal propagation robustness. Astrocyte swelling, observed in pathological conditions such as brain injury, stroke and epilepsy, results in an increased shaft width without altering node size. Our results suggest that such pathological alterations of the nanoscale morphology of astrocytes result in a decreased local Ca2+ activity, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neuronal stimuli, we predict that swelling hinders astrocytic signal propagation. Overall, this study highlights the impact of astrocyte nano-morphology on astrocyte activity at tripartite synapses, in health and disease.

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Population differences in the length and early-life dynamics of telomeres among European pied flycatchers Telomere length and shortening rate are increasingly used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between- population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the non-breeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north- south gradient (Finland, Estonia, England, and Spain) predicting negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from mid-latitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level. An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be up-regulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results indicate that pKFDV02 did not keep its promises as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

Key messagepPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes. Comparing genome-based estimates of relatedness for use in pedigree-based conservation management Researchers have long debated which estimator of relatedness best captures the degree of relationship between two individuals. In the genomics era, this debate continues, with relatedness estimates being sensitive to the methods used to generate markers, marker quality, and levels of diversity in sampled individuals. Here, we compare six commonly used genome-based relatedness estimators (kinship genetic distance (KGD), Wang Maximum Likelihood (TrioML), Queller and Goodnight (Rxy), Kinship INference for Genome-wide association studies (KING-robust), and Pairwise Relatedness (RAB), allele-sharing co-ancestry (AS)) across five species bred in captivity-including three birds and two mammals-with varying degrees of reliable pedigree data, using reduced-representation and whole genome resequencing data. Genome-based relatedness estimates varied widely across estimators, sequencing methods, and species, yet the most consistent results for known first order relationships were found using Rxy, RAB, and AS. However, AS was found to be less consistently correlated with known pedigree relatedness than either Rxy or RAB. Our combined results indicate there is not a single genome-based estimator that is ideal across different species and data types. To determine the most appropriate genome-based relatedness estimator for each new dataset, we recommend assessing the relative: (1) correlation of candidate estimators with known relationships in the pedigree and (2) precision of candidate estimators with known first-order relationships. These recommendations are broadly applicable to conservation breeding programs, particularly where genome-based estimates of relatedness can complement and complete poorly pedigreed populations. Given a growing interest in the application of wild pedigrees, our results and are also applicable to in-situ wildlife management. 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. If Research is Not the Evidence, What is it? The Lack of Research Citations in Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context. Effect of Adolescent Stress on Adult Morphine-Induced Behavioral Sensitization is Dependent Upon Genetic Background 1Deaths related to opioid use have skyrocketed in the United States, leading to a public health epidemic. Research has shown that both biological (genes) and environmental (stress) precursors are linked to opioid use. In particular, stress during adolescence - a critical period of frontal lobe development - influences the likelihood of abusing drugs. However, little is known about the biological mechanisms through which adolescent stress leads to long-term risk of opioid use, or whether genetic background moderates this response. Male and female C57BL/6J and BALB/cJ mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence and then tested for morphine locomotor sensitization or morphine consumption in adulthood. To examine possible mechanisms that underlie stress-induced changes in morphine behaviors, we assessed physiological changes in response to acute stress exposure and prefrontal cortex miRNA gene expression. Adolescent stress did not influence morphine sensitization or consumption in BALB/cJ animals, and there was limited evidence of stress effects in female C57BL/6J mice. In contrast, male C57BL/6J mice exposed to adolescent CVSS had blunted morphine sensitization compared to control animals; no differences were observed in the acute locomotor response to morphine administration or morphine consumption. Physiologically, C57BL/6J mice exposed to CVSS had an attenuated corticosterone recovery following an acute stressor and downregulation of twelve miRNA in the prefrontal cortex compared to control mice. The specificity of the effects for C57BL/6J versus BALB/cJ mice provides evidence of a gene by environmental interaction influencing opioid behaviors. Long-term differences in stress reactivity or miRNA expression suggests two possible biological mechanisms to evaluate in future research. The evolution of stomatal traits along the trajectory towards C4 photosynthesis C4 photosynthesis optimizes plants carbon and water relations, allowing high photosynthetic rate with low stomatal conductance. Stomata have long been believed as a part of C4 syndrome. However, it remains unclear how stomata traits evolved along the path from C3 to C4. Stomatal patterning was examined in Flaveria genus, a model for studying C4 evolution. Comparative, transgenic and semi-in-vitro experiments were used to study molecular basis that underlies stomatal traits along C4 evolution. Novel results: the evolution from C3 to C4 species through intermediate species is accompanied by a stepwise rather than an abrupt change in the stomatal traits. The initial change occurs near Type II and dramatic change occurs at the C4-like species. On the road to C4, stomata become less in number but bigger in size and changes in stomatal density dominates changes in maximum stomatal conductance (gsmax). The reduction of FSTOMAGEN expression underlies altered gsmax between Flaveria species with different photosynthetic pathways and likely occurs in other C4 lineages. Our study provides insight into the pattern, mechanism and role of stomatal evolution along the road towards C4. This work highlights the stomatal traits in the current C4 evolutionary model and the co-evolution of photosynthetic pathway and stomata. A cross biomes bacterial diversity shed light on ocean-atmosphere microbial transmission. Microbes are ubiquitous in the oceans and the atmosphere, playing essential roles in biogeochemical processes. The bio-exchanges between the two environments can provide important insights into microbial distribution and diversity but are still not well understood. We simultaneously surveyed the genomic diversity of airborne and marine bacterial communities across 15 000 kilometers in the Atlantic and Pacific oceans. Higher variability of microbial community composition was observed in the atmosphere than in the ocean surface waters. In addition, a greater similarity was observed between oceans than their overlaying atmosphere, and between atmospheric samples than with the ocean beneath. We additionally detected a higher coverage rate and relative abundance of marine bacteria in the Pacific atmosphere as compared to the Atlantic, while the dominant fraction in the Atlantic atmosphere was annotated as soil-associated bacteria. This study advances our understanding of microbial dispersion in the ocean, the atmosphere, and the exchange between them, as well as their potential impact on microbial composition, ecology, and biogeochemistry. Hierarchical autoencoder-based integration improves performance in multi-omics cancer survival models through soft modality selection With decreasing costs of high-throughput sequencing, more and more datasets providing omics profiles of cancer patients become available. Thus, novel survival analysis approaches integrating these differently sized and heterogeneous molecular and clinical groups of variables start being developed. Due to the difficulty of the task of multi-omics data integration, the Cox Proportional-Hazards (PH) model using clinical data has remained one of the best-performing techniques, barely outperformed by models using molecular data modalities. There is therefore a need for methods that can successfully perform multi-omics integration in survival analysis and outperform the clinical Cox PH model. Moreover, while certain deep learning methods have been shown to provide state-of-the-art accuracy of cancer survival prediction, most of them show no benefit or even decay in performance when integrating a larger number of modalities, further motivating a need to investigate how modality-specific representations should be integrated when using neural networks for multi-omics integration. We benchmarked multiple integration techniques for a neural network architecture, revealing that hierarchical autoencoder-based integration of modality-specific representations outperformed other methods such as max-pooling and was comparable with state-of-the-art statistical approaches for multi-omics integration. Further, we showed that the hierarchical autoencoder-based integration of modality-specific representations achieved increased performance through a soft modality selection mechanism, focusing on the most informative modalities for each cancer. We thus framed multiomics integration as a partial group-wise feature selection problem, highlighting that only those models performed well that could adequately weight important modalities in the presence of the high noise imposed by less important modalities. The Inflammatory Role of Milk Fat Globule Epidermal Growth Factor VIII in Angiotensin II Induced Arterial Remodeling BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown.

Methods and ResultsIn this study, 30-week-old MFG-E8 knock out (KO) and age-matched wild-type (WT) mice were infused with Ang II or saline. After infusion the with Ang II, the aortic molecular, cellular, and structural remodeling were observed in mice and compared to those infused with saline, but these effects were dependent on the expression of MFG-E8: (1) In the WT mice, Ang II infusion substantially increased intimal-medial thickness, elastic lamina degradation, collagen deposition, and the proliferation of VSMCs; in contrast, in the KO mice, these effects were significantly reduced; (2) In the WT mice, Ang II treatment significantly increased the activation and expression of MMP2, TGF-{beta}1, and its downstream signaling molecule p-SMAD2, and collagen type I production, however, in the KO mice, these molecular effects were significantly reduced; (3) In the WT mice, Ang II treatment increased inflammatory p-NF-{kappa}B p65, MCP1, TNF-, ICAM1, and VCAM1 molecular expression, while conversely, in the KO mice, no significant inflammatory changes were found; (4) Importantly, compared to untreated control mice with a wide range of age from 4-96 weeks, Ang II infused "younger" mice produced an "older" arterial inflammatory phenotype, which was alleviated by MFG-E8 deficiency.

ConclusionsMFG-E8 mediates Ang II associated arterial inflammatory remodeling. Targeting MFG-E8 is a novel molecular approach to curb adverse arterial remodeling during aging and hypertension.

CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Both Ang II and MFG-E8 increases are involved in proinflammatory arterial remodeling mediating the molecular, cellular and tissue events in aging and hypertension.
* MFG-E8 is essential for Ang II induced and age-associated adverse arterial remodeling via the increase of proinflammation, intimal medial thickening, elastin fragmentation, collagen deposition, and VSMC proliferation.


What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension. Multiscale mechanistic modelling of the host defense in invasiveaspergillosis reveals leukocyte activation and iron acquisition as driversof infection outcome Aspergillus species are ubiquitous environmental molds, the spores of which are inhaled daily by most humans. Immunocompromised hosts can develop an invasive infection resulting in high mortality. There is, therefore, a pressing need for host-centric therapeutics for this infection. To address this need, we created a multi-scale computational model of the infection, focused on its interaction with the innate immune system and iron, a critical nutrient for the pathogen. The model, parameterized using published data, was found to recapitulate a wide range of biological features and was experimentally validated in vivo. Conidial swelling was identified as critical in fungal strains with high growth, whereas the siderophore secretion rate is an essential prerequisite for establishment of the infection in low-growth strains. In immunocompetent hosts, high growth, high swelling probability, and impaired leukocyte activation lead to a high conidial germination rate. Similarly, in neutropenic mice, high fungal growth was achieved through synergy between high growth rate, high swelling probability, slow leukocyte activation, and high siderophore secretion. In summary, the model reveals a small set of parameters related to fungal growth, iron acquisition, and leukocyte activation as key determinants of the fate of the infection. Heterologous vaccination regimens with self-amplifying RNA and Adenoviral COVID vaccines induce robust immune responses in mice Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two dose heterologous vaccination regimens than single dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Environmental selection and epistasis in an empirical phenotype-environment-fitness landscape Fitness landscapes, mappings of genotype/phenotype to their effects on fitness, are invaluable concepts in evolutionary biochemistry. Though widely discussed, measurements of phenotype-fitness landscapes in proteins remain scarce. Here, we quantify all single mutational effects on fitness and phenotype of VIM-2 {beta}-lactamase across a 64-fold range of ampicillin concentrations. We then construct a phenotype-fitness landscape that takes variations in environmental selection pressure into account. We found that a simple, empirical landscape accurately models the ~39,000 mutational data points, suggesting the evolution of VIM-2 can be predicted based on the selection environment. Our landscape provides new quantitative knowledge on the evolution of the {beta}-lactamases and proteins in general, particularly their evolutionary dynamics under sub-inhibitory antibiotic concentrations, as well as the mechanisms and environmental dependence of nonspecific epistasis.

One Sentence SummaryAn empirical fitness landscape discloses the environmental dependence of mutational effects in VIM-2 {beta}-lactamase. Infection and transmission of ancestral SARS-CoV-2 and its alpha variant in pregnant white-tailed deer SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals. Nelfinavir induces cytotoxicity towards high-grade serous ovarian cancer cells, involving induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction of HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction of clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho- eIF2, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile. Comparative analysis of cattle breeds as satellite cell donors for cultured beef BackgroundCultured meat is a promising new field with the potential for considerable environmental and animal welfare benefits. One technological approach to cultured meat production utilises the proliferative and differentiative capacity of muscle-derived satellite cells (SCs) to produce large volumes of cultured muscle tissue from small biopsies of donor animals. Differing genotypes between cattle breeds lead to predictable phenotypic traits, resulting in breeds being favoured for their respective meat or milk production characteristics in the livestock industry. However, whilst these breeds show significant differences in muscle growth, it is unclear whether the physiological differences observed between them in vivo are reflected in differences in SC behaviour in vitro, particularly with respect to proliferation, differentiation and cellular longevity, and hence whether particular breeds might represent preferred SC donors for a cultured beef bioprocess.

ResultsComparing SCs isolated from five breeds (Belgian Blue, Holstein Friesian, Galloway, Limousin and Simmental), we found that the proliferation rates were largely unaffected by the donor breed. In contrast, potentially meaningful differences were observed in the kinetics and extent of myogenic differentiation. Furthermore, whilst differentiation dropped for all breeds with increasing population doublings (PDs), SCs from Belgian Blue and Limousin cattle showed significantly longer retention of differentiation capacity over long-term passaging.

ConclusionSCs from all breeds were able to proliferate and differentiate, although Limousin and (particularly) Belgian Blue cattle, both breeds commonly used for traditional meat production, may represent preferred donors for cultured beef production. Structure-Functional-Selectivity Relationship Studies on A-86929 Analogs and Small biaryl Fragments toward Discovery of biased D1 agonists Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through the D1- (D1R and D5R) and D2-class (D2R, D3R and D4R) of seven transmembrane receptors, and activates both G-protein- and {beta}-arrestin-dependent signaling pathways, to mediate its physiological effects. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinsons disease, or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side-effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling towards the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side-effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity-relationship (SAR) studies have embarked on discovering {beta}-arrestin-biased dopamine agonists, focused on D2 partial agonists, non-catechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R {beta}-arrestin biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold. Previously described and novel analogs of A-86929 were synthesized and screened in vitro for structure-functional-selectivity relationships (SFSR) studies to identify chemical motifs responsible for {beta}-arrestin biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein biased but none of them were exclusively arrestin-biased. Additionally, various catechol aryl fragments were designed and synthesized. Other compounds surveyed included hydroxyl and chloro analogs of dopamine to test for hydrogen bonding and ionic interactions. Some of these small molecular probes displayed weak bias towards the {beta}-arrestin pathway. Continued in-depth SFSR studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate discovery of potent and efficacious arrestin-biased dopamine receptor ligands. Atomic Force Microscopy Reveals Distinct Protofilament-scale Structural Dynamics in Depolymerizing Microtubule Arrays The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes, implicated in both the complete demolition and fine size control of microtubule-based arrays, are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here we use Atomic Force Microscopy (AFM) to image depolymerizing arrays at single microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-sized cytoskeletal structures.

SIGNIFICANCE STATEMENTOne cannot help but marvel at the precise organization of microtubule polymers in cellular structures such as the axoneme and the spindle. However, our understanding of the biochemical mechanisms that sculpt these arrays comes largely from in vitro experiments with a small number (one or two) of microtubules. This is somewhat akin to studying the architecture of multilane highways by studying one-lane streets. Here we directly visualize depolymerizing microtubule arrays at individual microtubule and protofilament resolution using Atomic Force Microscopy. Our results reveal differences in microtubule depolymerase activity and provide insights into how these differences in enzymatic activity on the nanometer scale can result in the differential remodeling of multi-microtubule arrays on the micron-length scale. Inhibitory proteins block substrate access to the membrane-embedded active site of Bacillus subtilis intramembrane metalloprotease SpoIVFB Intramembrane proteases function in numerous signaling pathways that impact health, but knowledge about regulation of intramembrane proteolysis is limited. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that BofA residues in and near a predicted transmembrane segment are required for SpoIVFB inhibition, and cross-linking experiments indicated that this transmembrane segment occupies the SpoIVFB active site cleft. SpoIVFA is also required for SpoIVFB inhibition. The inhibitory proteins block access of the substrate N-terminal Proregion to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a transmembrane segment and a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of intramembrane protease inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics. Effect of a low concentration in chloride, sodium and potassium on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout. Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity, and more specifically of low water concentrations in sodium, chloride and potassium, during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species.

Fish from the same commercial strain were raised either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting higher concentration in these elements. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation.

Here we show that low water salinity (i.e., low water concentration in chloride, sodium and potassium) negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggered delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (i.e., aquaporins) and ion (i.e., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the overall ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process.

HighlightsO_LILow water salinity impairs final oocyte maturation and egg quality in rainbow trout
C_LIO_LILow water salinity induces delayed ovulation and impaired oocyte hydration
C_LIO_LILow water salinity induces a dysregulation of several key ovulatory genes
C_LIO_LIMonitoring of final oocyte maturation can be performed using ultrasound staging
C_LI From GWAS Variant to Function: a Study of ~148,000 Variants for Blood Cell Traits. Genome-wide association studies (GWAS) have identified hundreds of thousands of genetic variants associated with complex diseases and traits. However, most variants are noncoding and not clearly linked to genes, making it challenging to interpret these GWAS signals. We present a systematic variant-to-function study, prioritizing the most likely functional elements of the genome for experimental follow-up, for >148,000 variants identified for hematological traits. Specifically, we developed VAMPIRE: Variant Annotation Method Pointing to Interesting Regulatory Effects, an interactive web application implemented in R Shiny (http://shiny.bios.unc.edu/vampire/). This tool efficiently integrates and displays information from multiple complementary sources, including epigenomic signatures from blood cell relevant tissues or cells, functional and conservation summary scores, variant impact on protein and gene expression, chromatin conformation information, as well as publicly available GWAS and phenome-wide association study (PheWAS) results. Leveraging data generated from independently performed functional validation experiments, we demonstrate that our prioritized variants, genes, or variant-gene links are significantly more likely to be experimentally validated. This study not only has important implications for systematic and efficient revelation of functional mechanisms underlying GWAS variants for hematological traits, but also provides a prototype that can be adapted to many other complex traits, paving the path for efficient variant to function (V2F) analyses. Feedforward regulatory logic underlies robustness of the specification-to-differentiation transition and fidelity of terminal cell fate during C. elegans endoderm development Development is driven by gene regulatory networks (GRNs) that progressively dictate specification and differentiation of cell fates. The architecture of GRNs directly determines the specificity and accuracy of developmental outcomes. We report here that the core regulatory circuitry for endoderm development in C. elegans is comprised of a recursive series of interlocked feedforward modules linking a cascade of six sequentially expressed GATA-type transcription factors. This structure results in a reiterated sequential redundancy, in which removal of a single factor or alternate factors in the cascade results in no, or a mild, effect on endoderm development and gut differentiation, while elimination of any two factors that are sequentially deployed in the cascade invariably results in a strong phenotype. The strength of the observed phenotypes is successfully predicted by a computational model based on the timing and levels of transcriptional states. The feedforward regulatory logic in the GRN appears to ensure timely onset of terminal differentiation genes and allows rapid and robust lockdown of cell fate during early embryogenesis. We further found that specification-to-differentiation transition is linked through a common regulator, the END-1 GATA factor that straddles the two processes. Finally, we revealed roles for key GATA factors in establishing spatial regulatory state domains by acting as transcriptional repressors that appear to define the boundaries of the digestive tract. Our findings support a comprehensive model of the core gene network that describes how robust endoderm development is achieved during C. elegans embryogenesis.

Graphic abstract

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org.highwire.dtl.DTLVardef@8515d6org.highwire.dtl.DTLVardef@15ca234org.highwire.dtl.DTLVardef@9d0fe7org.highwire.dtl.DTLVardef@17f28db_HPS_FORMAT_FIGEXP  M_FIG C_FIG Dynamic modulation of enhancer responsiveness by core promoter elements in living Drosophila embryo Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancers activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. Here, we provide evidence that each core promoter element dynamically alters "enhancer responsiveness" by changing functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. We also show that core promoters use the upstream TATA box as an amplifier of transcriptional bursting upon interaction with enhancers. Genome editing analysis of the pair-rule gene fushi tarazu further revealed that the endogenous TATA box and the downstream core promoter element are both essential for the correct expression and the function of key developmental genes in early embryos. We suggest that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development.