Dynamic interplay between a TonB dependent heme transporter and a TonB protein in a membrane environment The envelope of Gram-negative bacteria is composed of a double membrane separated by the periplasmic space. This organization imposes geometrical and distance constraints that are key for the mechanism of action of multicomponent systems spanning the envelope. However, consideration of all three compartments by experimental approaches is still elusive. Here we used the state-of-the-art molecular dynamics simulation in an Escherichia coli envelope model to obtain a dynamic view of molecular interactions between the outer membrane heme transporter HasR and the inner membrane TonB-like protein HasB. Their interaction allows the transfer of the inner membrane proton motive force derived energy to the transporter for heme internalization. The simulations which incorporate both membranes show the key role of periplasmic domains of both proteins, and their dynamics in the complex formation and stability. They revealed a previously unidentified atomic network of interactions, as well as the sequences of the interactions and their variations with the presence of external substrates. Experimental validation (mutations, phenotypic and in vitro assays) confirms the robustness of our approach and provides verification of the simulation-predicted interactions. Based on structural and sequence conservation, the network of interaction revealed in this study is expected to occur in other nutrient import systems. The integrative approach presented here is highly useful to study the dynamic interplay between components of any other bacterial transmembrane systems.
Absence of cardiolipin from the outer-leaflet of a mitochondrial inner-membrane mimic restricts Opa1-mediated fusion Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.
Detection and functional resolution of soluble multimeric immune complexes by a comprehensive FcγR reporter cell panel 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. Directed functional and structural connectivity in a large-scale model for the mouse cortex AO_SCPLOWBSTRACTC_SCPLOWInferring the structural connectivity from electrophysiological measurements is a fundamental challenge in systems neuroscience. Directed functional connectivity measures, such as the Generalized Partial Directed Coherence (GPDC), provide estimates of the causal influence between areas. However, the relation between causality estimates and structural connectivity is still not clear. We analyzed this problem by evaluating the effectiveness of GPDC to estimate the connectivity of a ground-truth, data-constrained computational model of a large-scale network model of the mouse cortex. The model contains 19 cortical areas comprised of spiking neurons, with areas connected by long-range projections with weights obtained from a tract-tracing cortical connectome. We show that GPDC values provide a reasonable estimate of structural connectivity, with an average Pearson correlation over simulations of 0.74. Moreover, even in a typical electrophysiological recording scenario containing five areas, the mean correlation was above 0.6. These results suggest that it may be possible to empirically estimate structural connectivity from functional connectivity even when detailed whole-brain recordings are not achievable. Protein scaffold-based multimerization of soluble ACE2 efficiently blocks SARS-CoV-2 infection in vitro and in vivo Soluble ACE2 (sACE2) decoy receptors are promising agents to inhibit SARS-CoV-2, as their efficiency is less likely to be affected by common escape mutations in viral proteins. However, their success may be limited by their relatively poor potency. To address this challenge, we developed a large decoy library of sACE2 fusion proteins, generated with several protease inhibitors or multimerization tags. Among these decoys, multimeric sACE2 consisting of SunTag or MoonTag systems, which were originally utilized for signal amplification or gene activation systems, were extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates. These novel sACE2 fusion proteins exhibited greater than 100-fold SARS-CoV-2 neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag in combination with a more potent version of sACE2, which has multiple point mutations for greater binding (v1), achieved near complete neutralization at a sub-nanomolar range, comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutant versions of Spike, alpha, beta, gamma or delta variants, were also neutralized efficiently with SunTag or MoonTag fused sACE2(v1). Finally, therapeutic treatment of sACE2(v1)-MoonTag provided protection against SARS-CoV-2 infection in an in vivo mouse model. Overall, we suggest that the superior activity of the sACE2-SunTag or sACE2-MoonTag fusions is due to the greater occupancy of the multimeric sACE2 receptors on Spike protein as compared to monomeric sACE2. Therefore, these highly potent multimeric sACE2 decoy receptors may offer a promising treatment approach against SARS-CoV-2 infections.

One Sentence SummaryMultimerization of sACE2 markedly enhanced the neutralization of SARS-CoV-2 by blocking multiple viral spike proteins simultaneously. The DC1 domain protein BINUCLEATE POLLEN is required for pollen development in Arabidopsis thaliana Development of the male gametophyte is a tightly regulated process that requires precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying the defective BNP allele failed to complete mitosis II and are impaired in pollen germination. By yeast two hybrid screening and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors we found NAC family transcriptional regulators Vascular plant One-Zinc finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP, as in their absence in pollen from bnp/BNP plants, VOZ1 nuclear localization resulted impaired. As voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have a role of scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology 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. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. DNA Strand Breaks and Gaps Direct Retroviral Integration Integration into a host genome is essential for retrovirus infection and is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and reverse transcribed (RT) copy DNA (cDNA). Previous studies demonstrated DNA site recognition limited intasome integration. Using single molecule Forster resonance energy transfer (smFRET), we show Prototype Foamy Virus (PFV) intasomes pause at DNA strand breaks and gaps. The break/gap discontinuities are similar to base excision repair (BER) lesion-processing intermediates, which affect retrovirus integration in vivo. Pausing targeted site-directed integration at the break/gap without inducing intasome conformational alterations. An 8-oxo-guanine lesion normally processes by BER and a G/T mismatch or a +T nucleotide insertion that induce flexibility or a bend in the DNA backbone did not promote intasome pausing or targeted integration. These results suggest that repair intermediates can modulate dynamic intasome-DNA interactions which target retroviral integration. 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. In vitro maturation of Toxoplasma gondii bradyzoites in human myotubes and their metabolomic characterization The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention. Genomic insights into longan evolution from a chromosome-level genome assembly and population genomics of longan accessions Longan (Dimocarpus longan) is a subtropical fruit best known for its nutritious fruit and regarded as a precious tonic and traditional medicine since ancient times. High-quality chromosome-scale genome assembly is valuable for functional genomic study and genetic improvement of longan. Here, we report a chromosome-level reference genome sequence for longan cultivar JDB with an assembled genome of 455.5 Mb in size anchored to fifteen chromosomes, representing a significant improvement of contiguity (contig N50=12.1 Mb, scaffold N50= 29.5 Mb) over a previous draft assembly. A total of 40,420 protein-coding genes were predicted in D. longan genome. Synteny analysis suggests longan shares the widespread gamma event with core eudicots, but has no other whole genome duplications. Comparative genomics showed that D. longan genome experienced significant expansions of gene families related to phenylpropanoid biosynthesis and UDP-glucosyltransferase. Deep genome sequencing analysis of longan cultivars identified longan biogeography as a major contributing factor for genetic diversity, and revealed a clear population admixture and introgression among cultivars of different geographic origins, postulating a likely migration trajectory of longan overall confirmed by existing historical records. Finally, genome-wide association studies (GWAS) of longan cultivars identified quantitative trait loci (QTL) for six different fruit quality traits and revealed a shared QTL containing three genes for total soluble solid and seed weight. The chromosome-level reference genome assembly, annotation and population genetic resource for D. longan will facilitate the molecular studies and breeding of desirable longan cultivars in the future. Higher-order effects, continuous species interactions, and trait evolution shape microbial spatial dynamics The assembly and maintenance of microbial diversity in natural communities, despite the abundance of toxin-based antagonistic interactions, presents major challenges for biological understanding. A common framework for investigating such antagonistic interactions involve cyclic dominance games with pairwise interactions. The incorporation of higher-order interactions in such models permits increased levels of microbial diversity, especially in communities where antibiotic producing, sensitive, and resistant strains co-exist. However, most such models involve a small number of discrete species, assume a notion of pure cyclic dominance, and focus on low mutation rate regimes, none of which well represents the highly interlinked, quickly evolving, and continuous nature of microbial phenotypic space. Here, we present an alternative vision of spatial dynamics for microbial communities based on antagonistic interactions--one in which a large number of species interact in continuous phenotypic space, are capable of rapid mutation, and engage in both direct and higher-order interactions mediated by production of and resistance to antibiotics. Focusing on toxin production, vulnerability, and inhibition among species, we observe highly divergent patterns of diversity and spatial community dynamics. We find that species interaction constraints (rather than mobility) best predict spatiotemporal disturbance regimes, whereas community formation time, mobility, and mutation size best explain patterns of diversity. We also report an intriguing relationship among community formation time, spatial disturbance regimes, and diversity dynamics. This relationship, which suggests that both higher-order interactions and rapid evolution are critical for the origin and maintenance of microbial diversity, has broad-ranging links to the maintenance of diversity in other systems.

Significance StatementPersistently diverse microbial communities are one of biologys great puzzles. Using a novel continuous trait space modeling framework that accommodates high mutation rates, elevated species richness, and direct and higher-order antagonistic species interactions, we find that two parameters characterizing mutation size and mobility best explain patterns of microbial diversity. Moreover, community formation time (the duration of the transient phase in community assembly) provides an unexpectedly clear guide to the diversity profiles of the resulting communities. These discoveries showcase how complex, antagonistic interactions mediated by the production of, inhibition of, and vulnerability to toxins (antibiotics) can shape microbial communities, allowing for extraordinarily high levels of diversity and temporal persistence. Differential fate of acellular vascular scaffolds in vivo involves macrophages and T-cell subsets Biological scaffold or implant is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortage in the clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix by potentially inducing immune responses after implantation. The current study explores an alternative route for the preparation of acellular scaffolds and explores the fate of the prepared scaffolds in a milieu of immune cells following implantation without using immunosuppressant. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs omentum) for transplantation, normal blood vascular scaffolds were implanted which was converted to acellular vascular scaffolds by in vivo natural decellularization at the end of 2 months of observation. We also prepared chemically decellularized acellular scaffolds from normal untreated blood vascular scaffolds using a cocktail of chemicals which was also similarly placed in subcutaneous and omentum sites. Here, we applied in-depth quantitative proteomics along with histology and image analysis to comprehensively describe and compare the proteome of the natural and chemically decellularized scaffold. Our data confirm that site-specific advantages exist in modulating the ECM and regulating the immune responses (macrophage and T cells) following implantation, which possibly led to the production of an acellular scaffold (natural decellularization) under in vivo conditions. The current approach opens up the possibility to create tailor-made acellular scaffolds to build functional blood vessels. In addition, the identification of different tissue sites facilitates differential immune response against the scaffolds. This study provides a rich resource aimed toward an enhanced mechanistic understanding to study immune responses under similar settings in the field of transplantation and regenerative medicine.

Impact statementThe development of a scaffold helps in the preparation of a functional organ in the clinics. In the current study, we prepared an acellular vascular scaffold by utilizing site specific tissue changes and vis-a-vis compared with a conventionally chemically prepared biological scaffold at genomic and protein level, which helped us to identify immunological trigger following implantation. The current study which was carried out without any immunosuppressive agents could help to establish (a) alternative strategies for preparing biological scaffolds as well as (b) implantable sites as potential bioreactors to circumvent any adverse immune reactions for acceptance of the scaffold/implant post implantation. Online single-cell data integration through projecting heterogeneous datasets into a common cell-embedding space Computational tools for integrative analyses of diverse single-cell experiments are facing formidable new challenges including dramatic increases in data scale, sample heterogeneity, and the need to informatively cross-reference new data with foundational datasets. Here, we present SCALEX, a deep-learning method that integrates single-cell data by projecting cells into a batch-invariant, common cell-embedding space in a truly online manner (i.e., without retraining the model). SCALEX substantially outperforms online iNMF and other state-of-the-art non-online integration methods on benchmark single-cell datasets of diverse modalities, (e.g., scRNA-seq, scATAC-seq), especially for datasets with partial overlaps, accurately aligning similar cell populations while retaining true biological differences. We showcase SCALEXs advantages by constructing continuously expandable single-cell atlases for human, mouse, and COVID-19 patients, each assembled from diverse data sources and growing with every new data. The online data integration capacity and superior performance makes SCALEX particularly appropriate for large-scale single-cell applications to build-upon previously hard-won scientific insights. Protein-coding potential of RNAs measured by potentially translated island scores Recent studies have identified numerous RNAs that are functionally both coding and noncoding. However, the sequence characteristics that determine bifunctionality remain largely unknown. In this study, we developed and tested a potentially translated island (PTI) score, defined as the occupancy of the longest open reading frame (ORF) among all putative ORFs. We found that this score correlated with translation, including noncoding RNAs. In bacteria and archaea, coding and noncoding transcripts had narrow distributions of high and low PTI scores, respectively, whereas those of eukaryotes showed relatively broader distributions, with considerable overlap between coding and noncoding transcripts. The extent of overlap positively and negatively correlated with the mutation rates of genomes and effective population sizes of species, respectively. These overlaps were significantly increased in threatened species. In macroevolution, the appearance of the nucleus and multicellularity seem to have influenced the overlap of PTI score distributions, so that the probability of the existence of bifunctional RNAs is increased in eukaryotes. In mammalian testes, we observed an enrichment of noncoding RNAs with high PTI scores, which are candidates for bifunctional RNAs. These results suggest that the decrease in population size and the emergence of testes in eukaryotic multicellular organisms allow for the stable existence of bifunctional RNAs, consequently increasing the probability of the birth of novel coding and non-coding RNAs. Effects of the social environment on movement-integrated habitat selection O_LIMovement links the distribution of habitats with the social environment of animals using those habitats; yet integrating movement, habitat selection, and socioecology remains an opportunity for further study.
C_LIO_LIHere, our objective was to disentangle the roles of habitat selection and social association as drivers of collective movement in a gregarious ungulate. To accomplish this objective, we (1) assessed whether socially familiar individuals form discrete social communities and whether social communities have high spatial, but not necessarily temporal, overlap; and (2) we modelled the relationship between collective movement and selection of foraging habitats using socially informed integrated step selection analysis.
C_LIO_LIWe used social network analysis to assign individuals to social communities and determine short and long-term social preference among individuals. Using integrated step selection functions (iSSF), we then modelled the effect of social processes, i.e., nearest neighbour distance and social preference, and movement behaviour on patterns of habitat selection.
C_LIO_LIBased on assignment of individuals to social communities and home range overlap analyses, individuals assorted into discrete social communities, and these communities had high spatial overlap. By unifying social network analysis with iSSF, we identified movement-dependent social association, where individuals foraged with more familiar individuals, but moved collectively with any between foraging patches.
C_LIO_LIOur study demonstrates that social behaviour and space use are inter-related based on spatial overlap of social communities and movement-dependent habitat selection. Movement, habitat selection, and social behaviour are linked in theory. Here, we put these concepts into practice to demonstrate that movement is the glue connecting individual habitat selection to the social environment.
C_LI Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model. The neurogenic fate of the hindbrain boundaries relies on Notch-dependent asymmetric cell divisions The generation of cell diversity in the central nervous system occurs during embryogenesis and requires a precise balance between cell proliferation, commitment to specific fates, and further neuronal differentiation. The cellular and molecular mechanisms regulating this balance in the embryonic brain are still poorly understood. Here we study how the neurogenic capacity in the embryonic hindbrain is spatiotemporally allocated, and how the fate and the growth of the hindbrain boundary cells are regulated. By generating a CRISPR-based knock-in zebrafish transgenic line to specifically label the hindbrain boundaries, we unveiled that boundary cells undergo a functional transition to become neurogenic during hindbrain segmentation concurrently as they maintain the progenitor cell pool. Boundary cells engaged in neurogenesis coinciding with the onset of Notch signaling, which triggered their asymmetrical cell division. Our findings reveal that distinct neurogenic phases take place during hindbrain growth and suggest that boundary cells contribute to refine the final number, identity, and proportion of neurons in the brain.

SUMMARYHindbrain boundary cells undergo a functional transition to become neurogenic concurrently as they are maintained as a progenitor cell pool. This involves a behavioral switch from symmetrically dividing progenitor cells to asymmetrically dividing progenitors, which depends on Notch-activity. 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. 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. Classification of Pacemaker Dynamics in the Mouse Intestine by Field Potential Microimaging The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals. Desalination and temperature increase will shift seasonal grazing patterns of invasive Gammarus tigrinus on charophytes Charophytes are a refuge for zooplankton and stabilize sediments, but they are also a food source for various animal species (water birds, fishes, invertebrates). Especially the introduction of new species, like Gammarus tigrinus, into the Baltic Sea led to yet not understood changes in the food web. Furthermore, future projections point to increased water temperatures at lowered salinity levels affecting species capacity to acclimatize to changing abiotic factors. In this study we investigated the influence of temperature and salinity on the grazing pressure of Gammarus tigrinus on two charophyte species: Chara aspera and Chara tomentosa. The grazing experiments were conducted in a full factorial design with the factors salinity (3 - 13 g kg-1), temperature (5 - 30 {degrees}C), and charophyte species. Grazing rates were determined as mass deviation within 48 hours considering biomass changes in the presence and absence of gammarids. Grazing rate were further used to calculate charophyte losses in two coastal lagoons with different salinity concentrations for recent and future time periods. The potential grazing peak of about 24 {degrees}C is not yet reached in these coastal waters but may be reached in the near future as shown by our future projection results. However, the temperature increase, and desalination will cause a shift in seasonal individual grazing patterns from summer to spring and autumn. Desalination and temperature increase can lead to a shift in optimal habitats for G. tigrinus in the future. Phenotypic heterogeneity driven by plasticity of the intermediate EMT state governs disease progression and metastasis in breast cancer The Epithelial-to-Mesenchymal Transition (EMT) is a developmental cellular program frequently coopted by cancer cells1 and is a key contributor to both heterogeneity in solid tumors2-4 and later stage chemo-resistance and metastasis5,6. Rather than being a switch from an epithelial to a mesenchymal state, increasing evidence points to the existence of intermediate EMT states, wherein cells co-express both epithelial and mesenchymal traits7-13. Multiple stable intermediate EMT states possessing unique characteristics exist across the EMT spectrum7,8,14,15, contributing to the complex heterogeneity of tumors to promote metastasis16. While much work has been carried out identifying and characterizing EMT-inducing transcription factors17-20, the transcriptional and epigenetic networks responsible for the stability and maintenance of the midpoints along the EMT spectrum are poorly defined. In addition, there are currently no approaches to identifying and quantifying intermediate EMT subpopulations within patient tumors to evaluate their prognostic significance. Using clonally isolated derivatives of the SUM149PT breast cancer cell line, we systematically interrogate how each EMT state independently contributes to heterogeneity and influences metastatic progression, uncovering the role of RUNX2 in stabilizing certain intermediate states. Using SUM149PT-derived tumors as a training set, we develop an entropy-based model to quantify phenotypic heterogeneity and EMT status. Remarkably, the majority of cell states captured in the SUM149PT model are represented in a selection of patient tumors, laying the foundation for quantification of epithelial-mesenchymal heterogeneity and understanding the role of the intermediate EMT state in tumor progression. 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. Neurological mechanism of sensory deficits after developmental exposure to non-dioxin-like polychlorinated biphenyls (PCBs) in zebrafish larvae The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to chemicals can result in neurobehavioral disorders. To uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits, we investigated the effects of the non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response using zebrafish as a vertebrate model. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. Morphological and biochemical data demonstrate that while exposure to PCB153 induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling is the dominant mechanism associated with motor movement. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and, with the startle response being critical for the survival of fish, to evolutionary adaptation in wildlife.