Genome annotation of Caenorhabditis briggsae by TEC-RED identifies new exons, paralogs, and conserved and novel operons The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying the 5 ends of expressed genes, we have discovered operons and paralogs. In summary, our analysis yielded 10,243 unique 5 end sequence tags with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. These genes included 14 that are exclusively trans-spliced in C. briggsae when compared with C. elegans orthologs. A major contribution of this study is the identification of 493 operons, of which two-thirds are fully supported by tags. In addition, two SL1-type operons were discovered. Interestingly, comparisons with C. elegans showed that only 40% of operons are conserved. Of the remaining operons, 73 are novel, including 12 that entirely lack orthologs in C. elegans. Further analysis revealed that four of the 12 novel operons are conserved in C. nigoni. Altogether, the work described here has significantly advanced our understanding of the C. briggsae system and serves as a rich resource to aid biological studies involving this species.
Phylogenetic analysis of molecular pathways in Tregs Suppressor Function Treg-mediated suppression of conventional T cells is a fundamental step in regulating the adaptive immune response. It is known that Treg first appeared in vertebrates. However, little is known about the of major suppression pathways mediated by Tregs. We employed artificial intelligence text mining system to highlight the suppression pathways currently known to be utilized by Tregs. Our system identified various pathways mediated by CTLA4, calcium signaling, NfkB and NFAT. We simultaneously employed detailed phylogenetic analysis including multiple sequence alignment, phylogenetic tree building, ancestral sequence reconstruction, neutrality tests and positive selection test to investigate the evolution of Treg mediated pathways. We found that CTLA4 first appeared in vertebrates possibly arising from an IGV containing protein in cartilaginous fish. Conversely, we found that Tregs repurposed ancient pathways such as Calcineurin and CAMP Response Element Modulator. Interestingly, these two pathways were highly conserved between vertebrates and lower invertebrates indicating conservation of function. Taken together, our research indicate that Tregs developed its regulatory systems through evolution in vertebrates as well as reusing conserved ancient regulatory systems that are related to the innate immune system.
Dynamics of SARS-CoV-2 mutations reveals regional-specificity and similar trends of N501 and High-Frequency mutation N501Y in different levels of control measures Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has spread globally, causing more than 161.5 million cases and 3.3 million deaths to date. Surveillance and monitoring of new mutations in the virus genome are crucial to our understanding of the adaptation of SARS-CoV-2. Moreover, how the temporal dynamics of these mutations is influenced by control measures and non-pharmaceutical interventions (NPIs) is poorly understood. Using 1 058 020 SARS-CoV-2 from sequenced COVID-19 cases from 98 countries (totaling 714 country-month combinations), we perform a normalization by COVID-19 cases to calculate the relative frequency of SARS-CoV-2 mutations and explore their dynamics over time. We found 115 mutations estimated to be present in more than 3 % of global COVID-19 cases and determined three types of mutation dynamics: High-Frequency, Medium-Frequency, and Low-Frequency. Classification of mutations based on temporal dynamics enable us to examine viral adaptation and evaluate the effects of implemented control measures in virus evolution during the pandemic. We showed that Medium-Frequency mutations are characterized by high prevalence in specific regions and/or in constant competition with other mutations in several regions. Finally, taking N501Y mutation as representative of High-Frequency mutations, we showed that level of control measure stringency negatively correlates with the effective reproduction number of SARS-CoV-2 with High-Frequency or not-High-Frequency and both follows similar trends in different levels of stringency. The Mitochondrial RNA Granule Modulates Manganese-Dependent Cell Toxicity Prolonged manganese exposure causes manganism, a neurodegenerative movement disorder. The identity of adaptive and non-adaptive cellular processes targeted by manganese remains mostly unexplored. Here we study mechanisms engaged by manganese in genetic cellular models known to increase susceptibility to manganese exposure, the plasma membrane manganese efflux transporter SLC30A10 and the mitochondrial Parkinsons gene PARK2. We found that SLC30A10 and PARK2 mutations as well as manganese exposure compromised the mitochondrial RNA granule as well as mitochondrial transcript processing. These RNA granule defects led to impaired assembly and function of the mitochondrial respiratory chain. Notably, cells that survived a cytotoxic manganese challenge had impaired RNA granule function, thus suggesting that this granule phenotype was adaptive. CRISPR gene editing of subunits of the mitochondrial RNA granule, FASTKD2 or DHX30, as well as pharmacological inhibition of mitochondrial transcription-translation, were protective rather than deleterious for survival of cells acutely exposed to manganese. Similarly, adult Drosophila mutants with defects in the mitochondrial RNA granule component scully were safeguarded from manganese-induced mortality. We conclude that the downregulation of the mitochondrial RNA granule function is a protective mechanism for acute metal toxicity.

Significance StatementMutations in the manganese efflux transporter SLC30A10 and the mitochondrial Parkinsons gene PARK2, cause neurodegeneration and increased susceptibility to toxic manganese exposure. Thus, molecular processes affected in both mutants could offer insight into fundamental mechanisms conferring susceptibility or resilience to environmental and genetic factors associated with neurodegeneration. Here we report that SLC30A10 and PARK2 mutations compromise an understudied structure, the mitochondrial RNA granule, which is required for processing polycistronic mitochondrial RNAs. Cells and Drosophila lacking mitochondrial RNA granule components were resistant to manganese exposure. We conclude that the downregulation of the mitochondrial RNA granule function is an adaptive mechanism for cells exposed to manganese. Modulation of P2X4 pore closure by magnesium,potassium, and ATP The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 {micro}s we have investigated how metal-complexed ATP stabilizes the channels open state and prevents its closing. We have identified two metal-binding sites, magnesium (Mg2+) and potassium (K+), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 resisted collapsing of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and we found evidence of pore collapse. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+. Neprosin belongs to a new family of glutamic peptidase family based on in silico evidence Neprosin was first discovered in the insectivorous tropical pitcher plants of Nepenthes species as a novel protease with prolyl endopeptidase (PEP) activity. Neprosin has two uncharacterized domains of neprosin activation peptide and neprosin. A previous study has shown neprosin activity in hydrolyzing proline-rich gliadin, a gluten component that triggers celiac disease. In this study, we performed in silico structure-function analysis to investigate the catalytic mechanism of neprosin. Neprosin sequences lack the catalytic triad and motifs of PEP family S9. Protein structures of neprosins from Nepenthes x ventrata (NvNpr) and N. rafflesiana (NrNpr1) were generated by ab initio methods and comparatively assessed to obtain high-quality models. Structural alignment of models to experimental structures in the Protein Data Bank (PDB) found a high structural similarity to glutamic peptidases. Further investigations reveal other resemblances to the glutamic peptidases with low optimum pH that activates the enzyme via autoproteolysis for maturation. Two highly conserved glutamic acid residues, which are stable according to the molecular dynamics simulation, can be found at the active site of the substrate cleft. Protein docking demonstrated that mature neprosins bind well with potent antigen I-gliadin at the putative active site. Taken together, neprosins represent a new glutamic peptidase family, with a putative catalytic dyad of two glutamic acids. This study illustrates a hypothetical enzymatic mechanism of the neprosin family and demonstrates the useful application of an accurate ab initio protein structure prediction in the structure-function study of a novel protein family. Towards a cumulative science of vocal markers of autism: a cross-linguistic meta-analysis-based investigation of acoustic markers in American and Danish autistic children Acoustic atypicalities in speech production are argued to be potential markers of clinical features in Autism Spectrum Disorder (ASD). A recent meta-analysis highlighted shortcomings in the field, in particular small sample sizes and study heterogeneity (Fusaroli et al., 2017). We showcase a cumulative (i.e., explicitly building on previous studies both conceptually and statistically) yet self-correcting (i.e., critically assessing the impact of cumulative statistical techniques) approach to prosody in ASD to overcome these issues.

We relied on the recommendations contained in the meta-analysis to build and analyze a cross-linguistic corpus of multiple speech productions in 77 autistic and 72 neurotypical children and adolescents (>1000 recordings in Danish and US English). We used meta-analytically informed and skeptical priors, with informed priors leading to more generalizable inference. We replicated findings of a minimal cross-linguistically reliable distinctive acoustic profile for ASD (higher pitch and longer pauses) with moderate effect sizes. We identified novel reliable differences between the two groups for normalized amplitude quotient, maxima dispersion quotient, and creakiness. However, the differences were small, and there is likely no one acoustic profile characterizing all autistic individuals. We identified reliable relations of acoustic features with individual differences (age, gender), and clinical features (speech rate and ADOS sub-scores).

Besides cumulatively building our understanding of acoustic atypicalities in ASD, the study shows how to use systematic reviews and meta-analyses to guide the design and analysis of follow-up studies. We indicate future directions: larger and more diverse cross-linguistic datasets, focus on heterogeneity, self-critical cumulative approaches and open science.

Lay SummaryAutistic individuals are reported to speak in distinctive ways. Distinctive vocal production can affect social interactions and social development and could represent a noninvasive way to support the assessment of ASD. We systematically checked whether acoustic atypicalities highlighted in previous articles could be actually found across multiple recordings and two languages. We find a minimal acoustic profile of ASD: higher pitch, longer pauses, increased hoarseness and creakiness of the voice. However, there is much individual variability (by age, sex, language, and clinical characteristics). This suggests that the search for one common "autistic voice" might be naive and more fine-grained approaches are needed. TeamTree analysis: a new approach to evaluate scientific production Advances in science and technology depend on the work of research teams and the publication of results through peer-reviewed articles representing a growing socio-economic resource. Current methods to mine the scientific literature regarding a field of interest focus on content, but the workforce credited by authorship remains largely unexplored, and appropriate measures of scientific production are debated. Here, a new bibliometric approach named TeamTree analysis is introduced that visualizes the development and composition of the workforce driving a field. A new citation-independent measure that scales with the H index estimates impact based on publication record, genealogical ties and collaborative connections. This author-centered approach complements existing tools to mine the scientific literature and to evaluate research across disciplines. A Novel Organoid Model of In Vitro Spermatogenesis Using Human Induced Pluripotent Stem Cells Infertility is thought to be caused by genetic mutations and dysfunction in the cellular niche where spermatogenesis takes place. An understanding of the specialized cellular processes which drive spermatogenesis is needed to develop treatments; however, the development of in vitro systems to study these cells has been hindered by our reliance on rarely available human testicular tissues for research. Human induced pluripotent stem cells (hiPSCs) can be used to derive human testicular-like cells, and thus provide an avenue for the development of in vitro testicular model systems. Therefore, this study set out to engineer a human testicular tissue model using hiPSCs for the first time. We demonstrate the ability of hiPSC-derived testicular cells to self-organize and mature into testicular-like tissues using organoid culture. Moreover, we show that hiPSC-derived testicular organoids promote testicular somatic cell maturation and spermatogenesis up to the post-meiotic spermatid stage. These hiPSC-derived testicular organoids have the potential to replace rarely available primary testicular tissues to further infertility research in an in vitro setting. A combined human in silico and CRISPR/Cas9-mediated in vivo zebrafish based approach for supporting gene target validation in early drug discovery The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy. The response network of HSP70 defines vulnerabilities in cancer cells with the inhibited proteasome Human neoplasias are often addicted to the proteasome machinery. However, cancers have evolved efficient response mechanisms to overcome proteasome inhibition with bortezomib and carfilzomib - drugs approved for multiple myeloma treatment. To understand these responses we investigated proteome changes upon the proteasome inhibition with carfilzomib - in multiple myeloma, normal fibroblasts, and cancers of lung, colon, and pancreas. A pathway-oriented siRNA screen based on the proteomics results showed that molecular chaperones, autophagy- and endocytosis-related proteins are cancer-specific vulnerabilities combined with carfilzomib. Targeting of HSPA1A/B (HSP70 family chaperones) most specifically sensitized cancer cells and patient-derived organoids to the proteasome inhibition. A high level of HSPA1A/B mRNA correlated with a low proteasome activity in cancer patient tissues and is a risk factor in cancer patients with a low proteasome expression. Mechanistically, HSPA1A/B governed autophagy, unfolded protein response, endocytic trafficking, and chaperoned the proteasome machinery, suppressing the effect of the proteasome inhibition, but did not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, downregulation of NRF1 most specifically decreased the viability of cancer cells with the inhibited proteasome and HSP70. 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. Toxicity Profile of Artesunate in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel. Antibacterial potency of Type VI amidase effector toxins is dependent on substrate topology and cellular context Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance (NMR) and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions. Neutralization of SARS-CoV-2 Omicron BA.2 by Therapeutic Monoclonal Antibodies Monoclonal antibody therapy for the treatment of SARS-CoV-2 infection has been highly successful in decreasing disease severity; however, the recent emergence of the heavily mutated Omicron variant has posed a challenge to this treatment strategy. The Omicron variant BA.1 has been found to evade neutralization by several of the therapeutic monoclonal antibodies authorized for emergency use, while Vir-7831 and a cocktail consisting of monoclonal antibodies AZD8895+AZD1061 retain significant neutralizing activity. A newly emerged variant, Omicron BA.2, containing some of the BA.1 mutations plus an additional 6 mutations and 3 deletions, 3 of which lie in the receptor binding domain, has been found to be spreading with increased transmissibility. We report here, using spike protein-pseudotyped lentiviruses, decreased neutralization of BA.2 by several therapeutic monoclonal antibodies but that the mixture of AZD8895+AZD1061 retained substantial neutralizing activity against BA.2. Climate warming changes synchrony of plants and pollinators Climate warming changes the phenology of many species. When interacting organisms respond differently, climate change may disrupt their interactions and affect the stability of ecosystems. Here, we used GBIF occurrence records to examine phenology trends in plants and their associated insect pollinators in Germany since the 1980s. We found strong phenological advances in plants, but differences in the extent of shifts among pollinator groups. The temporal trends in plant and insect phenologies were generally associated with interannual temperature variation, and thus likely driven by climate change. When examining the synchrony of species-level plant-pollinator interactions, their temporal trends differed among pollinator groups. Overall, plant-pollinator interactions become more synchronized, mainly since the phenology of plants responded more strongly to climate change than that of the pollinators. However, if the observed trends continue, many interactions may become more asynchronous again in the future. Our study suggests that climate change affects the phenologies of both plants and insects, and that it also influences the synchrony of plant-pollinator interactions. Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic andtargetable tissue states Glioblastoma is an aggressive diffusely infiltrating neoplasm that spreads beyond surgical resection margins, where it intermingles with non-neoplastic brain cells. This complex microenvironment harbouring infiltrating glioma and non-neoplastic brain cells is the origin of tumor recurrence. Thus, understanding the cellular and molecular features of the glioma microenvironment is therapeutically and prognostically important. Here, we used single-nucleus RNA sequencing (snRNAseq) of primary and recurrent glioma to define three compositional  tissue-states rooted in cohabitation of cell-types and transcriptional states. These comprise a state featuring A) abundant normal brain microenvironment cells, B) reactive/inflamed brain microenvironment, and C) cellular/proliferative tumor. All these states exhibited variable degrees of infiltration by glioma cells. Spatial transcriptomics confirmed that the cell-types and transcriptomics states which compositionally cohabitate tissue states also colocalize in space. Tissue states are clinically significant because they correlate with radiographic, histopathologic, and prognostic features. We found that enrichment of tissue state B signature correlated with shorter survival. Importantly, we found that our compositionally defined tissue states are enriched in distinct metabolic pathways. One such pathway is fatty acid biosynthesis, which was enriched in tissue state B - a state enriched in recurrent glioblastoma and composed of astrocyte-like/mesenchymal glioma cells, reactive astrocytes resembling those seen in neurodegeneration, and monocyte-like myeloid cells. We showed that treating acute slices of GBM with a fatty acid synthesis inhibitor is sufficient to deplete the transcriptional signature of tissue state B. Our findings define a novel compositional approach to the glioma-infiltrated tissue which allows us to discover prognostic and targetable features, paving the way to new mechanistic and therapeutic discoveries. A novel Boolean network inference strategy to model early hematopoiesis aging Hematopoietic stem cell (HSC) aging is a multifactorial event that leads to changes in HSC properties and function. These changes are intrinsically coordinated and affect the early hematopoiesis, involving hematopoietic stem and progenitor cells (HSPCs). The objective of this work is to better understand the mechanisms and factors controlling these changes. We have therefore developed an original strategy to construct a Boolean network of genes explaining the priming and homeostasis of HSCs (graphical abstract). Based on our previous scRNA-seq data, we performed an exhaustive analysis of the transcriptional network and identified active transcription modules or regulons along the differentiation trajectory of selected HSPC states. This global view of transcriptional regulation led us to focus on 15 components, 13 selected TFs (Tal1, Fli1, Gata2, Gata1, Zfpm1, Egr1, Junb, Ikzf1, Myc, Cebpa, Bclaf1, Klf1, Spi1) and 2 complexes regulating the ability of HSC to cycle (CDK4/6 - Cyclin D and CIP/KIP). We then defined the connections controlling the differentiation dynamics of HSC states and constructed an influence graph between the TFs involved in the dynamics by mixing observations from our scRNA-seq data and knowledge from the literature. Then, using answer set programming (ASP) and in silico perturbation analysis, we obtained a Boolean model which is the solution of a Boolean satisfiability problem. Finally, perturbation of the model based on age-related changes revealed important regulations, such as the overactivation of Egr1 and Junb or the loss of Cebpa activation by Gata2, which were found to be relevant for the myeloid bias of aged HSC. Our work shows the efficiency of the combination of manual and systematic methods to elaborate a Boolean model. The developed strategy led to the proposal of new regulatory mechanisms underlying the differentiation bias of aged HSCs, explaining the decreased transcriptional priming of HSCs to all mature cell types except megakaryocytes. 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. Do males know? Evidence-driven rainmaking mating strategy by male bonobos to meet the fertile window of females Female bonobos exhibit prolonged receptivity, one suggested function of which is to lower male mating competition. However, it is questionable whether easier access to receptive females can reduce male-male competition, given the exclusive nature of male reproductive success. We tested whether males could determine the fertile phase of females. We found that ovulation probability predicted male mating effort. High-ranking males copulated with females with higher fertility, and male-male agonistic interactions increased when there were fertile females in the party. When there were multiple females with maximal swelling, males concentrated their mating effort on females with an older infant whose maximal swelling started earlier, and they continued mating efforts until detumescence (rainmaking). These findings suggest that male bonobos distinguish between fertile and non-fertile phases of females and that having more receptive females in the party does not reduce male-male competition for fertile females.

TeaserMales use the rainmakers rule to meet the periovulatory phase of the female bonobo for better reproductive success. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. FGF8-mediated signaling regulates tooth developmental pace during odontogenesis The developing human and mouse teeth constitute an ideal model system to study the regulatory mechanism underlying organ growth control due to the fact that their teeth share highly conserved and well-characterized developmental processes and their developmental tempo vary notably. In the current study, we manipulated heterogenous recombination between human and mouse dental tissues and demonstrate that the dental mesenchyme dominates the tooth developmental tempo and FGF8 could be a critical player during this developmental process. Forced activation of FGF8 signaling in the dental mesenchyme of mice promoted cell proliferation, prevented cell apoptosis via p38 and perhaps PI3K-Akt intracellular signaling, and impelled the transition of the cell cycle from G1- to S-phase in the tooth germ, resulting in the slowdown of the tooth developmental pace. Our results provide compelling evidence that extrinsic signals can profoundly affect tooth developmental tempo and the dental mesenchymal FGF8 could be a pivotal factor in controlling developmental pace in a non-cell-autonomous manner during mammalian odontogenesis. 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. Utility of SARS-CoV-2 infection models using in vitro and in vivo ACE2-lentivirus transduction SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intranasal hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lungs. RNA-Seq analyses illustrated that the model involves an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. Intranasal hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mice lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

AUTHOR SUMMARYSARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research. Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin BackgroundEvolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation at many enhancers, distal regulatory elements that control spatio-temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific, code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high.

ResultsWe present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that is able to predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Then, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity and liver enhancers associated with liver regeneration have a stronger tendency than the general population to have predicted lineage-specific open chromatin.

ConclusionThe framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements. State-dependent network interactions differentially gate sensory input at the motor and command neuron level in Caenorhabditis elegans Neural responses are influenced by both external stimuli and internal network states. While network states have been linked to behavioral and stimulus states, little is known about how sensory inputs are filtered by whole-brain activity to affect motor and command neurons. Here, we recorded whole-brain activity of Caenorhabditis elegans experiencing bacterial food stimuli, and modeled how sensory inputs affect motor and command neurons in a network state-dependent manner. First, we classified active neurons into six functional clusters: two sensory neuron clusters (ON, OFF), and four motor/command neuron clusters (AVA, RME, SMDD, SMDV). Using encoding models, we found that ON and OFF sensory neurons that respond to onset and removal of bacteria, respectively, employ different adaptation strategies. Next, we used decoding models to show that bacterial onset and removal differentially drive AVA and RME cluster activity. To explore state-dependent effects on AVA and RME clusters, we developed a model that identified network states and fitted submodels for each state to predict how each of the six functional clusters drive AVA and RME cluster activity. We also identified network states in which AVA and RME clusters were either largely unperturbed by or receptive to bacterial sensory input. Furthermore, this model allowed us to disentangle the state-dependent contributions of stimulus timescales and bacterial content to neural activity. Collectively, we present an interpretable approach for modeling network dynamics that goes beyond implication of neurons in particular states, and moves toward explicitly dissecting how neural populations work together to produce state dependence.

Significance StatementA major function of the brain is to transform sensory information into behavior. As the first receiver of sensory input, sensory neuron activity is often most correlated with stimulus features. However, this high-fidelity representation of sensory input becomes diluted as it travels to downstream neurons, where sensory information is integrated with network activity. By the time sensory information reaches motor neurons, it is often difficult to dissociate the influence of sensory input from the influence of network activity. Here, we describe a method that is fully interpretable such that we can show how neural populations on a whole-brain scale interact to produce network states. From there, we can attribute motor neuron activity to network history and sensory input.