An End-to-End Deep Learning Speech Coding and Denoising Strategy for Cochlear Implants Cochlear implant (CI) users struggle to understand speech in noisy conditions. To address this problem, we propose a deep learning speech denoising sound coding strategy that estimates the CI electric stimulation patterns out of the raw audio data captured by the micro-phone, performing end-to-end CI processing. To estimate the relative denoising performance differences between various approaches, we compared this technique to a classic Wiener filter and to a convTasNet. Speech enhancement performance was assessed by means of signal-to-noise-ratio improvement and the short-time objective speech intelligibility measure. Additionally, 5 CI users were evaluated for speech intelligibility in noise to assess the potential benefits of each algorithm. Our results show that the proposed method is capable of replacing a CI sound coding strategy while preserving its general use for every listener and performing speech enhancement in noisy environments, without sacrificing algorithmic latency.
Genomically Mined Acoustic Reporter Genes Enable In Vivo Monitoring of Tumors and Tumor-Homing Bacteria A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is the difficulty of visualizing the function of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 {micro}m. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging in the in vivo context. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics, identified through a phylogenetic screen of candidate gene clusters from diverse bacteria and archaea. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue, and stable long-term expression. We demonstrate the utility of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing probiotic bacteria, revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells noninvasively in living subjects.
Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. Critical re-evaluation of the slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

Experimental ApproachFunctional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA, leading to wrong estimates of operational efficacy and affinity of an agonist. In contrast, functional analysis of OMA with Hill coefficient as a slope factor has not this flaw.

Key ResultsFitting of the OMA with Black et al. slope factor to concentration-response curves of several models (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) gives wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to concentration-response curves of these several models gives correct estimates of operational efficacy and affinity.

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias. Interpretable deep learning model reveals subsequences of various functions for long non-coding RNA identification Long non-coding RNAs (lncRNAs) play crucial roles in many biological processes and are implicated in several diseases. With the next-generation sequencing technologies, substantial un-annotated transcripts have been discovered. Classifying unannotated transcripts using biological experiments is more time-consuming and expensive than computational approaches. Several tools for identifying long non-coding RNAs are available. These tools, however, did not explain which features in their tools contributed to the prediction results. Here, we present Xlnc1DCNN, a tool for distinguishing long non-coding RNAs (lncRNAs) from protein-coding transcripts (PCTs) using a one-dimensional convolutional neural network with prediction explanations. The evaluation results of the human test set showed that Xlnc1DCNN outperformed other state-of-the-art tools in terms of accuracy and F1-score. The explanation results revealed that lncRNA transcripts were mainly identified as sequences with no conserved regions or with a region of transmembrane helix while protein-coding transcripts were mostly classified by conserved protein domains or families. The explanation results also conveyed the probably inconsistent annotations among the public databases, lncRNA transcripts which contain protein domains or families, as well as protein-coding transcripts which are nonsense-mediated decay or processed transcripts. Xlnc1DCNN is freely available at https://github.com/cucpbioinfo/Xlnc1DCNN. Using total RNA quality metrics for time since deposition estimates in degrading bloodstains Determining the time since deposition (TSD) of bloodstains would provide forensic scientists with critical information regarding the timeline of the events involving bloodshed. The physicochemical changes occurring to biomolecules as a bloodstain degrades can be used to approximate the TSD of bloodstains. Our study aims to quantify the timewise degradation trends and temperature dependence found in total RNA from bloodstains without the use of amplification, expanding the scope of the RNA TSD research which has previously targeted mRNA molecules. Whole bovine blood was stored in plastic microcentrifuge tubes at 21{degrees}C or 4{degrees}C and tested over different timepoints spanning one week. Nine RNA metrics were visually assessed and quantified using linear and mixed models; the RNA Integrity Number equivalent (RINe) and the DV200 demonstrated strong negative trends over time and statistical independence. The RINe model fit was high (R2 = 0.60), and while including the biological replicate as a random effect increased the fit for all RNA metrics, no significant differences were found between biological replicates stored at the same temperature for the RINe and DV200 metrics. Importantly, this suggests that these standardized metrics can likely be directly compared between scenarios and individuals, with DV200 having an inflection point at [~]28 hrs. This study provides a novel approach for blood TSD estimates, producing metrics that are not affected by inter-individual variation and improving our understanding of the rapid degradation occurring in bloodstains.

HIGHLIGHTSO_LIAmplification-free analysis of total RNA in degrading bloodstains.
C_LIO_LIShort-term RNA degradation assessment using high-resolution size measurements.
C_LIO_LITotal RNA quality and quantity metrics were assessed across a one-week.
C_LIO_LITotal RNA quality metrics demonstrated the strongest timewise trends.
C_LIO_LIBiological replicates produced similar results for RNA quality metrics.
C_LI "Adopt a Microorganism" methodology enhances the discourse richness of high school students in both hybrid learning and remote education The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Effects of substrate on shrimp growth, water quality and bacterial community in the biofloc system nursing Penaeus vannamei postlarvae This study aimed to investigate the effects of substrate on water quality, shrimp growth and bacterial community in the biofloc system with a salinity of 5{per thousand}. Two treatments, biofloc system with (sB) or without (nB) addition of elastic solid packing filler (nylon) as substrate, were set up. Penaeus vannamei postlarvae (PL, [~] stage 15) were stocked at a density of 4000 PL m-3 in each treatment with triplicates for a 28-days culture experiment, taking glucose as carbon source (C:N 15:1). Results showed that the survival rate (96.3{+/-}3.6%), FCR (0.76{+/-}0.06) and productivity (1.54{+/-}0.12 kg m-3) in sB treatment were significantly better than those in nB treatment (81.0{+/-}7.1%, 0.98{+/-}0.08 and 1.14{+/-}0.09 kg m-3, P<0.05). All water parameters were in the recommended ranges. Substrate showed significant effect on TAN, TSS, turbidity, biofloc volume, pH and carbonate alkalinity (P < 0.05). Actinobacteria (4.0-22.7%), Bacteroidetes (10.4-33.5%), Firmicutes (0.2-11.2%), Planctomycetes (4.0-14.9%) and Proteobacteria (29.4-59.0%) were the most dominant phyla for both treatments. However, the bacterial community in sB treatment showed to be significantly different from that in nB treatment (Jaccard distance 0.94{+/-}0.01, P=0.001). Substrate showed significant effects on Shannon, Heip, Pielou and Simpson index, as well as relative abundances of Actinobacteria, Bacteroidetes and Planctomycetes (P < 0.05). The results suggested that addition of substrate affected the shrimp growth, water quality and bacterial community in the biofloc system nursing P. vannamei PL with a 5{per thousand} salinity. The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. The H4K20 demethylase DPY-21 regulates thedynamics of condensin DC binding Condensin is a multi-subunit SMC complex that binds to and compacts chromosomes. Here we addressed the regulation of condensin binding dynamics using C. elegans condensin DC, which represses X chromosomes in hermaphrodites for dosage compensation. We established fluorescence recovery after photobleaching (FRAP) using the SMC4 homolog DPY-27 and showed that a well-characterized ATPase mutation abolishes its binding. Next, we performed FRAP in the background of several chromatin modifier mutants that cause varying degrees of X-chromosome derepression. The greatest effect was in a null mutant of the H4K20me2 demethylase DPY-21, where the mobile fraction of condensin DC reduced from [~]30% to 10%. In contrast, a catalytic mutant of dpy-21 did not regulate condensin DC mobility. Hi-C data in the dpy-21 null mutant showed little change compared to wild type, uncoupling Hi-C measured long-range DNA contacts from transcriptional repression of the X chromosomes. Together, our results indicate that DPY-21 has a non-catalytic role in regulating the dynamics of condensin DC binding, which is important for transcription repression. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. Expansion of mutation-driven haematopoietic clones is associated with insulin resistance and low HDL-cholesterol in individuals with obesity AimsHaematopoietic clones caused by somatic mutations with [&ge;]2% variant allele frequency (VAF), known as clonal haematopoiesis of indeterminate potential (CHIP), increase with age and have been linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones are also associated with adverse clinical outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes, and to examine the development of clones over time in relation to age and metabolic dysregulation over up to 20 years in individuals with obesity.

Methods and ResultsWe used an ultrasensitive single-molecule molecular inversion probe sequencing assay to identify clonal haematopoiesis driver mutations (CHDMs) in blood samples from individuals with obesity from the Swedish Obese Subjects study. In a single-timepoint dataset with samples from 1050 individuals, we identified 273 candidate CHDMs in 216 individuals, with VAF ranging from 0.01% to 31.15% and CHDM prevalence and clone sizes increasing with age. Longitudinal analysis over 20 years in CHDM-positive samples from 40 individuals showed that small clones can grow over time and become CHIP. VAF increased on average by 7% (range -4% to 27%) per year. Rate of clone growth was positively associated with insulin resistance (R=0.40, P=0.025) and low circulating levels of high-density lipoprotein-cholesterol (HDL-C) (R=-0.68, P=1.74E-05).

ConclusionOur results show that haematopoietic clones can be detected and monitored before they become CHIP and indicate that insulin resistance and low HDL-C, well-established cardiovascular risk factors, are associated with clonal expansion in individuals with obesity.

Translational perspectivesClonal haematopoiesis-driver mutations are somatic mutations in haematopoietic stem cells that lead to clones detectable in peripheral blood. Haematopoietic clones with a variant allele frequency (VAF) [&ge;]2%, known as clonal haematopoiesis of indeterminate potential (CHIP), are recognized as an independent cardiovascular risk factor. Here, we show that smaller clones are prevalent, and also correlate with age. Our longitudinal observations in individuals with obesity over 20 years showed that more than half of all clone-positive individuals show growing clones and clones with VAF <2% can grow and become CHIP. Importantly, clone growth was accelerated in individuals with insulin resistance and low high-density lipoprotein-cholesterol (HDL-C).

Translational outlook 1: Haematopoietic clones can be detected and monitored before they become CHIP.

Translational outlook 2: The association between insulin resistance and low HDL-C with growth of haematopoietic clones opens the possibility that treatments improving metabolism, such as weight loss, may reduce growth of clones and thereby cardiovascular risk.

One Sentence SummaryIn obesity, the growth rate of mutation-driven haematopoietic clones increased with insulin resistance and low HDL-C, both known risk factors for cardiovascular disease. Long-term translocation explains population genetic structure of a recreationally fished iconic species in Japan: Combining current knowledge with reanalysis Ayu (Plecoglossus altivelis altivelis) is an important freshwater fisheries resource and popular recreational fishing species in Japan, lives for only one year, and has a single breeding season. To supplement increased recreational fishing demand for this species, huge numbers of wild-born landlocked juveniles have been translocated from Lake Biwa into most Japanese rivers for more than 100 generations. Hatchery-reared juveniles born from captive-reared parents for more than 30 generations have also been extensively released. Previous studies have reported that landlocked and amphidromous forms of Ayu easily hybridise, but survival of landlocked larvae could be very low in seawater, leading to a general consensus among scientists, hatchery managers, and commercial and recreational fishers that the reproductive success of landlocked Ayu is very low, or even 0 in translocated rivers. Despite this, limited information exists regarding the reproductive success of landlocked Ayu in translocated rivers, and no study has evaluated the effects of translocation on population structure. Demonstrating that hybridisation occurs between the two forms is central to future management and conservation of this specie. To address this issue, a comprehensive literature search was undertaken, and three published genetic data sets are analysed. Analyses provide strong evidence for very high gene flow between populations, but population structure has been retained in several regions, and several populations are nested. Allele frequencies are similar in amphidromous and landlocked forms. Genetic diversity is homogeneous in amphidromous populations. Bayesian admixture analysis infers widespread introgression in Japanese rivers, with a mean introgression proportion of 24 {+/-} 8%. Maximum likelihood admixture graphs detect two migration events from Lake Biwa to anadromous populations. Analyses consistently indicate that hybridisation between translocated landlocked juveniles and native amphidromous Ayu occurs throughout Japanese rivers. 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. Thermal modulation of Zebrafish exploratory statistics reveals constraints on individual behavioral variability BackgroundVariability is a hallmark of animal behavior. It contributes to survival by endowing individuals and populations with the capacity to adapt to ever-changing environmental conditions. Intra-individual variability is thought to reflect both endogenous and exogenous modulations of the neural dynamics of the central nervous system. However, how variability is internally regulated and modulated by external cues remains elusive. Here we address this question by analyzing the statistics of spontaneous exploration of freely swimming zebrafish larvae, and by probing how these locomotor patterns are impacted when changing the water temperatures within an ethologically relevant range.

ResultsWe show that, for this simple animal model, five short-term kinematic parameters - interbout interval, turn amplitude, travelled distance, turn probability and orientational flipping rate - together control the long-term exploratory dynamics. We establish that the bath temperature consistently impacts the means of these parameters, but leave their pairwise covariance unchanged. These results indicate that the temperature merely controls the sampling statistics within a well-defined kinematic space delineated by this robust statistical structure. At a given temperature, individual animals explore the behavioral space over a timescale of tens of minutes, suggestive of a slow internal state modulation that could be externally biased through the bath temperature. By combining these various observations into a minimal stochastic model of navigation, we show that this thermal modulation of locomotor kinematics results in a thermophobic behavior, complementing direct gradient-sensing mechanisms.

ConclusionsThis study establishes the existence of a well-defined locomotor space accessible to zebrafish larvae during spontaneous exploration, and quantifies self-generated modulation of locomotor patterns. Intra-individual variability reflects a slow diffusive-like probing of this space by the animal. The bath temperature in turn restricts the sampling statistics to sub-regions, endowing the animal with basic thermophobicity. This study suggests that in Zebrafish, as well as in other ectothermic animals, ambient temperature could be used to efficiently manipulate internal states in a simple and ethological way. Enhancing CDK4/6 inhibitor therapy for medulloblastoma using nanoparticle delivery and scRNA-seq-guided combination with sapanisertib CDK4/6 inhibitors hold promise for brain tumor treatment, but efficacy has been limited by recurrence in both preclinical models and clinical trials. To address recurrence, we tested a nanoparticle formulation of the CDK4/6 inhibitor palbociclib (POx-palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma. We then analyzed medulloblastomas in mice receiving palbociclib treatment, and compared the efficacy of combining palbociclib with specific inhibitors suggested by our analysis. POx-Palbo showed reduced toxicity compared to conventional palbociclib, was tolerable in parenteral administration, improved CNS pharmacokinetics, and extended survival of mice with medulloblastoma. Recurrence, however, remained problematic as fractions of tumor cells proliferated during therapy. ScRNA-seq identified a gene expression pattern unique to proliferating medulloblastoma cells in POx-Palbo-treated mice, marked by up-regulation of the glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes. Reduced mTORC1 signaling, suggested by ribosomal suppression in POx-Palbo-treated tumors was confirmed by decreased 4EBP1 phosphorylation (p4EBP1). Further reducing mTORC1 activity by combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects, with increased suppression of both pRB and p4EBP1, and prolonged mouse survival compared to either agent alone. In contrast, targeting cell cycle progression by combining POx-Palbo with the SHH-pathway inhibitor vismodegib, or with the replication-targeting agents gemcitabine or etoposide, failed to enhance efficacy. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment, and identify POx-palbo plus sapanisertib as effective combinatorial therapy for SHH medulloblastoma. This combination may be appropriate for testing in patients with recurrence, who need new options. Chromosome counting in the mouse zygote using low-invasive super-resolution live-cell imaging In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse (Slc:ICR) embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the forty chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse embryos. The methodology presented here may provide useful information for assisted reproductive technologies, such as those used in livestock animals/humans, as a technique for assessing the chromosomal integrity of embryos prior to transfer.

Impact StatementLow-toxic super-resolution observation enables chromosome counting in preimplantation embryos without cell collection. Symbiodiniaceae conduct under natural bleaching stress during advanced gametogenesis stages of a mesophotic coral The mesophotic coral Alveopora allingi from the northern Gulf of Eilat/Aqaba, Red Sea, is affected by year-round partial coral-bleaching events. During these events, the migration of Symbiodiniaceae takes place from the coral-host mesoglea to the developed oocytes in bleached parts of colonies of A. allingi but not in the non-bleached parts. Additionally, these oocytes are abnormal, missing part of the structural material of the peripheral areas and are also significantly larger in the bleached areas of the colonies. Hence, we suggest a parasitic behavior of the symbionts or a commensalism relationship which enhance symbionts needs during bleaching periods and may boost the gametogenesis development in these corals. We propose that evolutionarily, this behavior may greatly contribute to the symbiont community survival throughout the bleaching period, and it can also be beneficial for the hosts persistence and adaptation to bleaching through the acquisition of a specific symbiont community following the bleaching event. An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress. Mobile elements harbouring heavy metal and bacitracin resistance cassettes are common among Listeria monocytogenes persisting on dairy farms Listeria monocytogenes is a food-borne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements of Listeria inhabiting dairy farms. We performed a SNP-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes that persistent on the farms for [&ge;] 6 months. We found that prophages and other mobile genetic elements were on average more numerous among isolates in persistent than nonpersistent clades, and demonstrated that resistance genes against bacitracin, arsenic and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons and a novel prophage harbouring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of Enterococci, indicative of recent transfer between these genera. Finally, we demonstrated that the CRISPR-cas IIa system and a type II restriction-modification system were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry.

ImportanceAnimal derived raw materials are an important source of L. monocytogenes for the food industry. Knowledge of the factors contributing to the pathogens transmission and persistence on farms are essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favourable phenotypes, such as resilience against biocides and thermal stress. Understanding the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to rise the novel resistance phenotypes. Distinct neural codes in primate Hippocampus and Lateral Prefrontal Cortex during associative learning in virtual environments The hippocampus (HPC) and the lateral prefrontal cortex (LPFC) are two cortical areas of the primate brain deemed essential to cognition. Here we hypothesize that the codes mediating neuronal communication in HPC and LPFC microcircuits have distinctively evolved to serve plasticity and memory function at different spatiotemporal scales. We used a virtual reality task in which animals navigated through a maze using a joystick and selected one of two targets in the arms of the maze according to a learned context-color rule. We found that neurons and neuronal populations in both regions encode similar information about the task. Moreover, we demonstrate that many HPC neurons concentrate spikes into bursts, whereas most layer II/III LPFC neurons sparsely distribute spikes over time. As the animals learned the task HPC neurons, but not LPFC neurons, increased their burst rate as a function of performance. When integrating spike rates over short intervals, HPC neuronal ensembles reached maximum decoded information with fewer neurons than LPFC ensembles. Our results show that during associative learning HPC principal cells concentrate spikes in bursts enabling temporal summation and fast synaptic plasticity in small populations of neurons and ultimately facilitating rapid encoding of associative memories. On the other hand, layers II/III LPFC pyramidal cells fire spikes more sparsely distributed in time and over a larger number of neurons. The latter would facilitate broadcasting of signals loaded in short term memory across neuronal populations without necessarily triggering fast synaptic plasticity. Immune complex solubility and size govern Fc-gamma receptor responses Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings. Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 M blebbistatin (BB, an excitation-contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput. Plant inositol-phosphate-glycans and a fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection In mammals, insulin is involved in controlling blood glucose levels and its role in modulating immunity is being more and more documented. This hormone promotes the release of inositolphosphate glycans (IPG) which act as mediators. In plants, one IG has already been identified in plant culture cells (Smith and Fry, 1999; Smith et al., 1999) but, to our knowledge, no IPG have been yet identified. Here, we discovered 7 IPG that are accumulated upon Arabidopsis thaliana-Botrytis cinerea interaction, concomitantly with oligogalacturonides and a fucosylated xyloglucan oligosaccharide. Further structural characterization showed that they come from the hydrolysis of polar heads of Serie A to H glycosylinositol phosphorylceramides presumably via a phospholipase C activity. Taken together with the emerging role of insulin as immune regulator, these results question the role of IPG as damage associated molecular pattern both in animal and plant kingdoms. Directed evolution of angiotensin-converting enzyme 2 (ACE2) peptidase activity profiles for therapeutic applications Angiotensin Converting-Enzyme 2 (ACE2) is currently being investigated for its ability to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases, but its broad substrate scope and diverse physiological roles limit its potential as a therapeutic agent. In this work we engineer ACE2 variants with enhanced hydrolytic activity and specificity toward Angiotensin-II (Ang-II) that may lead to new therapeutic candidates with improved efficacy and reduced off-target side effects. We established a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants with improved Ang-II activity and specificity relative to the off-target peptide substrates Apelin-13 and Angiotensin-I (Ang-I). We screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371 and Y510) that can be mutated to enhance ACE2s activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wildtype ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat), a sixfold diminished catalytic efficiency (kcat/Km) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes more Ang-II than wildtype ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts. Light activation of Orange Carotenoid Protein reveals initial C8'-C7' double bond trans/cis isomerization Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation1,2. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows carotenoid trans/cis isomerization at the C7-C8double bond and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven cis/trans isomerization that recovers to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. The isomerization and its transient nature are confirmed in OCP crystals and solution by FTIR and UV/Vis spectroscopy. This study reveals the isomerization of the carotenoid and subsequent thermal structural reactions as the basis of OCP photoreception. Understanding and potentially controlling the OCP dynamics offers the prospect of novel applications in biomass engineering3 as well as in optogenetics and bioimaging.