JACKIE: Fast enumeration of genomic single- and multi-copy target sites and their off-targets for CRISPR and other engineered nuclease systems ZFP-, TALE-, and CRISPR-based methods for genome, epigenome editing and imaging have provided powerful tools to interrogate functions of genomes. Targeting sequence design is vital to the success of these experiments. While existing design software mainly focus on designing target sequence for specific elements, we report here the implementation of JACKIE (Jackie and Alberts Comprehensive K-mer Instances Enumerator), a suite of software for enumerating all single- and multi-copy sites in the genome that can be incorporated for genome-scale designs as well as loaded onto genome browsers alongside other tracks for convenient web-based graphic-user-interface (GUI)-enabled design. We also implement fast algorithms to identify sequence neighborhoods or off-target counts of targeting sequences so that designs with low probability of off-target can be identified among millions of design sequences in reasonable time. We demonstrate the application of JACKIE-designed CRISPR site clusters for genome imaging.
Vocal markers of autism: assessing the generalizability of machine learning models Machine learning (ML) approaches show increasing promise in their ability to identify vocal markers of autism. Nonetheless, it is unclear to what extent such markers generalize to new speech samples collected e.g., using a different speech task or in a different language. In this paper, we systematically assess the generalizability of ML findings across a variety of contexts.

We train promising published ML models of vocal markers of autism on novel cross-linguistic datasets following a rigorous pipeline to minimize overfitting, including cross-validated training and ensemble models. We test the generalizability of the models by testing them on i) different participants from the same study, performing the same task; ii) the same participants, performing a different (but similar) task; iii) a different study with participants speaking a different language, performing the same type of task.

While model performance is similar to previously published findings when trained and tested on data from the same study (out-of-sample performance), there is considerable variance between studies. Crucially, the models do not generalize well to different, though similar, tasks and not at all to new languages. The ML pipeline is openly shared.

Generalizability of ML models of vocal markers of autism is an issue. We outline three recommendations for strategies researchers could take to be more explicit about generalizability and improve it in future studies.

LAY SUMMARYMachine learning approaches promise to be able to identify autism from voice only. These models underestimate how diverse the contexts in which we speak are, how diverse the languages used are and how diverse autistic voices are. Machine learning approaches need to be more careful in defining their limits and generalizability.
Chromosome fusion affects genetic diversity and evolutionary turnover of functional loci, but consistently depends on chromosome size Major changes in chromosome number and structure are linked to a series of evolutionary phenomena, including intrinsic barriers to gene flow or suppression of recombination due to chromosomal rearrangements. However, chromosome rearrangements can also affect the fundamental dynamics of molecular evolution within populations by changing relationships between linked loci and altering rates of recombination. Here, we build chromosome-level assembly Eueides isabella and, together with the chromosome-level assembly of Dryas iulia, examine the evolutionary consequences of multiple chromosome fusions in Heliconius butterflies. These assemblies pinpoint fusion points on 10 of the 21 autosomal chromosomes and reveal striking differences in the characteristics of fused and unfused chromosomes. The ten smallest autosomes in D. iulia and E. isabella, which have each fused to a longer chromosome in Heliconius, have higher repeat and GC content, and longer introns than predicted by their chromosome length. Following fusion, these characteristics change to become more in line with chromosome length. The fusions also led to reduced diversity, which likely reflects increased background selection and selection against introgression between diverging populations, following a reduction in per-base recombination rate. We further show that chromosome size and fusion impact turnover rates of functional loci at a macroevolutionary scale. Together these results provide further evidence that chromosome fusion in Heliconius likely had dramatic effects on population level processes shaping rates of neutral and adaptive divergence. These effects may have impacted patterns of diversification in Heliconius, a classic example of an adaptive radiation. Transcriptional and Functional Activity of Hemangiosarcoma Support Bone Marrow Nurse Cell Ontogeny Hemangiosarcoma and angiosarcoma are soft tissue sarcomas of malignant blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs that support the niche, enabling progression of canine hemangiosarcoma and human angiosarcoma. Here, we show that the transcriptional landscape of canine hemangiosarcoma and human angiosarcoma included comparable angiogenic and inflammatory programs. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells, a trait that was not observed in xenografts from canine osteosarcoma and lymphoma. In some cases, the xenografted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. We did not uncover a definitive transmissible etiology, but our data indicate that transcriptional programs of hemangiosarcoma cells resemble those of hematopoietic nurse cells, and these malignant cells support expansion and differentiation of human hematopoietic progenitors. We conclude that canine hemangiosarcoma, and possibly human angiosarcoma, originate from nurse cells that make up the stromal bone marrow niche, and that these cells may also support the growth of hematopoietic tumors. C. elegans PEZO-1 is a Mechanosensitive Ion Channel Involved in Food Sensation PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remain unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms. 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. Global analysis of multi-mutants to improveprotein function The identification of amino acid substitutions that both enhance the stability and function of a protein is a key challenge in protein engineering. Technological advances have enabled assaying thousands of protein variants in a single high-throughput experiment, and more recent studies use such data in protein engineering. We present a Global Multi-Mutant Analysis (GMMA) that exploits the presence of multiply-substituted variants to identify individual amino acid substitutions that are beneficial for the stability and function across a large library of protein variants. We have applied GMMA to >54,000 variants of green fluorescent protein (GFP), each with known fluorescence output, and each carrying 1-15 amino acid substitutions. The GMMA method achieves a good fit to the data while being analytically transparent. The six top-ranking substitutions are demonstrated to progressively enhance GFP and in general, our analysis recovers nearly all the substitutions previously reported to be beneficial for GFP folding and function, only using a single experiment as input.

Significance StatementProtein engineering is carried out to improve proteins for practical applications by changing one or more amino acid residues in a protein. We present a method termed global multi-mutant analysis (GMMA) that helps solve two problems in protein engineering. First, because many proteins are already highly optimized, it can be difficult to identify individual variants that further improve function. Second, while it is possible to combine variants with small individual effects, such approaches may be hampered by non-additivity. GMMA identifies combinable effects of single substitutions from a large set of variants each carrying multiple substitutions. We demonstrate the approach on a set of 54,000 variants of green fluorescent protein and identify many enhancing single-substitutions from a single experiment. Hnf1b-CreER causes efficient recombination of a Rosa26-RFP reporter in duct and islet δ cells The Hnf1b-CreERT2 BAC transgenic (Tg(Hnf1b-cre/ERT2)1Jfer) has been used extensively to trace the progeny of pancreatic ducts in developmental, regeneration, or cancer models. Hnf1b-CreERT2 transgenics have been used to show that cells from that form a duct-like plexus in the embryonic pancreas are bipotent duct-endocrine progenitors, whereas adult mouse duct cells are not a common source of {beta} cells in various regenerative settings. The interpretation of such genetic lineage tracing studies is critically dependent on a correct understanding of the cell type specificity of recombinase activity with each reporter system. We have re-examined the performance of Hnf1b-CreERT2 with a Rosa26-RFP reporter transgene. This showed inducible recombination of up to 96% adult duct cells, a much higher efficiency than previously used reporter transgenes. Despite this high duct-cell excision, recombination in  and {beta} cells remained very low, similar to previously used reporters. However, nearly half of somatostatin-expressing {delta} cells showed reporter activation, which was due to Cre expression in {delta} cells rather than to duct to {delta} cell conversions. The high recombination efficiency in duct cells indicates that the Hnf1b-CreERT2 model can be useful for both ductal fate mapping and genetic inactivation studies. The recombination in {delta} cells does not modify the interpretation of studies that failed to show duct conversions to other cell types, but needs to be considered if this model is used in studies that aim to modify the plasticity of pancreatic duct cells. 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. Full-length Genome of a Ogataea polymorpha strain CBS4732 ura3Δ reveals large duplicated segments in subtelomeric regions BackgroundCurrently, methylotrophic yeasts (e.g., Pichia pastoris, Ogataea polymorpha, and Candida boindii) are subjects of intense genomics studies in basic research and industrial applications. In the genus Ogataea, most research is focused on three basic O. polymorpha strains--CBS4732, NCYC495, and DL-1. However, the relationship between CBS4732, NCYC495, and DL-1 remains unclear, as the genomic differences between them have not be exactly determined without their high-quality complete genomes. As a nutritionally deficient mutant derived from CBS4732, the O. polymorpha strain CBS4732 ura3{Delta} (named HU-11) is being used for high-yield production of several important proteins or peptides. HU-11 has the same reference genome as CBS4732 (noted as HU-11/CBS4732), because the only genomic difference between them is a 5-bp insertion.

ResultsIn the present study, we have assembled the full-length genome of O. polymorpha HU-11/CBS4732 using high-depth PacBio and Illumina data. Long terminal repeat (LTR) retrotransposons, rDNA, 5 and 3 telomeric, subtelomeric, low complexity and other repeat regions were curated to improve the genome quality. Particularly, we detected large duplicated segments (LDSs) in the subtelomeric regions and exactly determined all the structural variations (SVs) between CBS4732 and NCYC495.

New findings mainly include(1) the genomic differences between HU-11/CBS4732 and NCYC495 include single nucleotide polymorphisms, small insertions and deletions, and only three SVs; (2) six genes were incorporated into CBS4732 from Cyberlindnera jadinii by horizontal gene transfer and may bring HU-11/CBS4732 new biological functions or physiological properties; (3) many recombination events may have occurred on chromosome 4 and 5 of CBS4732 and NCYC495 ancestors and two large segments were acquired by CBS4732 and NCYC495 from chromosome 6 and C. jadinii during recombination, respectively; and (4) the genome expansion in methylotrophic yeasts is mainly driven by large segment duplication in subtelomeric regions.

ConclusionsThe present study preliminarily revealed the complex relationship between CBS4732, NCYC495, and DL-1. The new findings provide new opportunities for in-depth understanding of genome evolution in methylotrophic yeasts and lay the foundations for the industrial applications of O. polymorpha CBS4732, NCYC495, DL-1, and their derivative strains. The full-length genome of the O. polymorpha strain HU-11/CBS4732 should be included into the NCBI RefSeq database for future studies of Ogataea spp.. Untangling the complexity of priority effects in multispecies communities The history of species immigration can dictate how species interact in local communities, thereby causing historical contingency in community assembly. Since immigration history is rarely known, these historical influences, or priority effects, pose a major challenge in predicting community assembly. Here, we provide a graph-based, non-parametric, theoretical framework for understanding the predictability of community assembly as affected by priority effects. To develop this frame-work, we first show that the diversity of possible priority effects increases super-exponentially with the number of species. We then point out that, despite this diversity, the consequences of priority effects for multispecies communities can be classified into four basic types, each of which reduces community predictability: alternative stable states, alternative transient paths, compositional cycles, and the lack of escapes from compositional cycles to stable states. Using a neural network, we show that this classification of priority effects enables accurate explanation of community predictability, particularly when each species immigrates repeatedly. We also demonstrate the empirical utility of our theoretical framework by applying it to two experimentally derived assembly graphs of algal and ciliate communities. Based on these analyses, we discuss how the framework proposed here can help guide experimental investigation of the predictability of history-dependent community assembly. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. A parametric logistic equation with light flux and medium concentration for cultivation planning of microalgae Microalgae are considered to be promising producers of bioactive chemicals, feeds, and fuels from carbon dioxide by photosynthesis. Thus, the prediction of microalgal growth profiles is important for the planning of cost-effective and sustainable cultivation-harvest cycles. This paper proposes a mathematical model capable of predicting the effect of light flux into culture and medium concentration on the growth profiles of microalgae by incorporating these growth-limiting factors into the logistic equation. The specific form of the equation is derived based on the experimentally measured growth profiles of Monoraphidium sp., a microalgal strain isolated by the authors, under 16 conditions consisting of the combinations of incident light fluxes into culture and initial medium concentrations. Using a cross-validation method, it is shown that the proposed model has the ability to predict necessary incident light flux into culture and initial medium concentration for harvesting target biomass at target time. Finally, the model-guided cultivation planning is performed and is evaluated by comparing the result with the experimental data. RAD51AP1 and RAD54 underpin two distinct RAD51-dependent routes of DNA damage repair via homologous recombination Homologous recombination (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory sub-pathways.

In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that deletion of both RAD51AP1 and RAD54 synergistically sensitizes human cancer cell lines to treatment with a Poly(adenosine 5 s-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which stalls the progression of DNA replication forks. We infer that HR-directed anti-cancer treatment modalities shall consider this intra-pathway functional overlap, and we hypothesize that in cancerous cells the simultaneous inactivation of both RAD54 and RAD51AP1 will accentuate tumor kill. Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics. Somatic uniparental disomy mitigates the most damaging EFL1 allele combination in Shwachman-Diamond syndrome Shwachman-Diamond syndrome (SDS; OMIM: #260400) is caused by variants in SBDS (Shwachman-Bodian-Diamond syndrome gene), which encodes a protein that plays an important role in ribosome assembly. Recent reports suggest that recessive variants in EFL1 are also responsible for SDS. However, the precise genetic mechanism that leads to EFL1-induced SDS remains incompletely understood. Here we present three unrelated Korean SDS patients that carry biallelic pathogenic variants in EFL1 with biased allele frequencies, resulting from a bone marrow-specific somatic uniparental disomy (UPD) in chromosome 15. The recombination events generated cells that were homozygous for the relatively milder variant, allowing for the evasion of catastrophic physiological consequences. Still, the milder EFL1 variant was solely able to impair 80S ribosome assembly and induce SDS features in cell line, zebrafish, and mouse models. The loss of EFL1 resulted in a pronounced inhibition of terminal oligo-pyrimidine element-containing ribosomal protein transcript 80S assembly. Therefore, we propose a more accurate pathogenesis mechanism of EFL1 dysfunction that eventually leads to aberrant translational control and ribosomopathy. Chromatin remodelling by INO80 at promoter proximal pause sites promotes premature termination of mRNA synthesis How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process. Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies. Behavioural analysis of swarming mosquitoes reveals higher hearing sensitivity than previously measured with electrophysiology methods Mosquitoes of many species mate in station-keeping swarms. Mating chases ensue as soon as a male detects the flight tones of a female with his auditory organs. Previous studies of hearing thresholds have mainly used electrophysiological methods that prevent the mosquito from flying naturally. The main aim of this study was to quantify behaviourally the sound-level threshold at which males can hear females. Free-flying male Anopheles coluzzii were released in a large arena (~2 m high x 2 m x 1 m) with a conspicuous object on the ground that stimulates swarming behaviour. Males were exposed to a range of natural and synthetic played-back sounds of female flight. We monitored the responses of males and their distance to the speaker by recording changes in their wingbeat frequency and angular speed. We show that the mean male behavioural threshold of particle-velocity hearing lies between 13-20 dB SVL (95%-CI). A conservative estimate of 20 dB SVL (i.e., < 0.5 m/s particle velocity) is already 12 to 26 dB lower than most of the published electrophysiological measurements from the Johnstons organ. In addition, we suggest that 1) the first harmonic of female flight-sound is sufficient for males to detect her presence, 2) males respond with a greater amplitude to single-female sounds than to the sound of a group of females and 3) the response of males to the playback of the flight sound of a live female is the same as that of a recorded sound of constant frequency and amplitude. Water levels primarily drive variation in photosynthesis and nutrient use of scrub Red Mangroves in the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades. Impairment of SARS-CoV-2 spike-glycoprotein maturation and fusion-activity by nitazoxanide: an effect independent of spike variants emergence SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion-glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike-variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike biogenesis, hampering its maturation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. 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. Reinforcement Learning and Bayesian Inference Provide Complementary Models for the Unique Advantage of Adolescents in Stochastic Reversal During adolescence, youth venture out, explore the wider world, and are challenged to learn how to navigate novel and uncertain environments. We investigated whether adolescents are uniquely adapted to this transition, compared to younger children and adults. In a stochastic, volatile reversal-learning task with a sample of 291 participants aged 8-30, we found that adolescents outperformed both younger and older participants. We developed two independent cognitive models, based on Reinforcement learning (RL) and Bayesian inference (BI). The RL parameter for learning from negative outcomes and the BI parameters specifying participants mental models peaked closest to optimal in adolescents, suggesting a central role in adolescent cognitive processing. By contrast, persistence and noise parameters improved monotonously with age. We distilled the insights of RL and BI using principal component analysis and found that three shared components interacted to form the adolescent performance peak: adult-like behavioral quality, child-like time scales, and developmentally-unique processing of positive feedback. This research highlights adolescence as a neurodevelopmental window that may be specifically adapted for volatile and uncertain environments. It also shows how detailed insights can be gleaned by using cognitive models in new ways. Brain endothelial STING1 activation by Plasmodium-sequestered heme promotes cerebral malaria via type I IFN response. Cerebral malaria (CM) is a life-threatening form of Plasmodium falciparum infection caused by brain inflammation. Brain endothelium dysfunction is a hallmark of CM pathology, which is also associated with the activation of the type I interferon (IFN) inflammatory pathway. The molecular triggers and sensors eliciting brain type I IFN cellular responses during CM remain largely unknown. We herein identified the stimulator of interferon response cGAMP interactor 1 (STING1) as the key innate immune sensor that induces Ifn{beta}1 transcription in the brain of mice infected with Plasmodium berghei (Pba). This STING1/IFN{beta}-mediated response increases brain CXCL10 governing the extent of brain leucocyte infiltration and blood-brain barrier (BBB) breakdown, and determining CM lethality. The critical role of brain endothelial cells (BECs) in fueling type I IFN-driven brain inflammation was demonstrated in brain endothelial-specific IFN{beta}-reporter and STING1-deficient Pba-infected mice, which are significantly protected from CM lethality. Moreover, extracellular particles (EPs) released from Pba-infected erythrocytes activated STING1-dependent type I IFN response in BECs, a response requiring intracellular acidification. Fractionation of the EPs enabled us to identify a defined fraction carrying hemoglobin degradation remnants that activates STING1/IFN{beta} in the brain endothelium, a process correlated with heme content. Notably, stimulation of STING1-deficient BECs with heme, docking experiments and in vitro binding assays unveiled that heme is a putative STING1 ligand. This work shows that heme resultant from the parasite heterotrophic activity operates as an alarmin triggering brain endothelial inflammatory responses via STING1/IFN{beta}/CXCL10 axis crucial to CM pathogenesis and lethality.

SignificanceCM results from loss of blood-brain endothelial barrier function caused by unrestrained inflammatory response in the natural course of infection by Plasmodium parasites. However, the role of brain endothelium in triggering inflammatory mechanisms is still undetermined. We found that the innate immune sensor STING1 is crucial for production of IFN{beta} in brain endothelial cells in Plasmodium-infected mice. This in turn stimulates CXCL10-mediated recruitment of leukocytes and subsequent brain inflammation and tissue damage. We identified within extracellular particles released from Plasmodium-infected erythrocytes, a fraction containing products of hemoglobin degradation, namely heme, which we show can bind STING1. Our results unravel a new angle of CM pathogenesis: heme contained in particles triggers the STING/IFN{beta}/CXCL10 axis in brain endothelial cells. Raman Signal Denoising using Fully Convolutional Encoder Decoder Network Raman spectroscopy is a vibrational method that gives molecular information rapidly and non-invasively. Despite its advantages, the weak intensity of Raman spectroscopy leads to low-quality signals, particularly with tissue samples. The requirement of high exposure times makes Raman a time-consuming process and diminishes its non-invasive property while studying living tissues. Novel denoising techniques using convolutional neural networks (CNN) have achieved remarkable results in image processing. Here, we propose a similar approach for noise reduction for the Raman spectra acquired with 10x lower exposure times. In this work, we developed fully convolutional encoder-decoder architecture (FCED) and trained them with noisy Raman signals. The results demonstrate that our model is superior (p-value < 0.0001) to the conventional denoising techniques such as the Savitzky-Golay filter and wavelet denoising. Improvement in the signal-to-noise ratio values ranges from 20% to 80%, depending on the initial signal-to-noise ratio. Thus, we proved that tissue analysis could be done in a shorter time without any need for instrumental enhancement.