Growth and division mode plasticity is dependent on cell density in marine-derived black yeasts The diversity and ecological contribution of the fungus kingdom in the marine environment remain under-studied. A recent survey in the Atlantic (Woods Hole, MA, USA) brought to light the diversity and unique biological features of marine fungi. The study revealed that black yeast species undergo an unconventional cell division cycle, which has not been documented in conventional model yeast species such as Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast). The prevalence of this unusual property is unknown. Here, I collected and identified 65 marine fungi species across 40 genera from the surface ocean water, sediment, and the surface of macroalgae (seaweeds) in the Pacific (Sugashima, Toba, Japan). The Sugashima collection largely did not overlap with the Woods Hole collection and included several unidentifiable species, further illustrating the diversity of marine fungi. Three black yeast species were isolated, two of which were commonly found in Woods Hole (Aureobasidium pullulans, Hortaea werneckii). Surprisingly, their cell division mode was dependent on cell density, and the previously reported unconventional division mode was reproduced only at a certain cell density. For all three black yeast species, cells underwent filamentous growth with septations at low cell density and immediately formed buds at high cell density. At intermediate cell density, two black yeasts (H. werneckii and an unidentifiable species) showed rod cells undergoing septation at the cell equator. In contrast, all eight budding yeast species showed a consistent division pattern regardless of cell density. This study suggests the plastic nature of the growth/division mode of marine-derived black yeast.
Reticular Dysgenesis-associated Adenylate Kinase 2 deficiency causes failure of myelopoiesis through disordered purine metabolism Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency that affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, lymphopenia, and deafness. The disease is caused by biallelic loss of function in mitochondrial Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of AMP to ADP, as substrate for ATP synthesis. Accordingly, declining oxidative phosphorylation has been postulated as the driver of disease pathology. The mechanistic basis, however, remains incompletely understood. Single cell RNA-sequencing of patient bone marrow cells implicated altered RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings, we developed a disease model based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. We found that AK2-deficient myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels of the purine nucleotide precursor IMP, decreased cellular RNA content, ribosome subunit expression, protein synthesis and a profoundly hypo-proliferative phenotype. The rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the disease phenotype, pointing to AMP catabolism as a possible metabolic adaptation to mitigate AMP-mediated toxicity. Inducing an adenosine disequilibrium in control cells produced a similar myeloid maturation defect.

This study shows that AK2 deficiency globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism. AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity may contribute to the failure of myelopoiesis in Reticular Dysgenesis.
Human readaptation outpaces loss of antibiotic resistance in livestock-associated MRSA Mobile genetic elements (MGEs) are agents of horizontal gene transfer in bacteria, but can also be vertically inherited by daughter cells. Establishing the dynamics that led to contemporary patterns of MGEs in bacterial genomes is central to predicting the emergence and evolution of novel and resistant pathogens. Here we develop existing methods to fully characterise the evolutionary history of MGEs in methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex (CC) 398. CC398 is the dominant MRSA in European livestock, and a growing cause of human infections. We reconstruct MGE dynamics using a collection of 1,180 CC398 genomes, sampled from livestock and humans, over 27 years. We find that the emergence of livestock-associated CC398 coincided with the acquisition of a Tn916 transposon carrying a tetracycline resistance gene, which has been stably vertically inherited for 57 years. This was followed by the acquisition of a type V SCCmec that carries methicillin, tetracycline and heavy metal resistance genes, which has been maintained by vertical and horizontal transmission for 35 years, with occasional truncations and replacements with type IV SCCmec. In contrast, a class of prophages that carry a human immune evasion gene cluster, that are largely absent from livestock-associated CC398, have been repeatedly gained and lost in both human- and livestock-associated CC398. These contrasting dynamics mean that when livestock-associated MRSA infects humans, adaptation to the human host outpaces loss of antibiotic resistance. In addition, stable inheritance means that antibiotic resistance in livestock-associated MRSA may persist despite ongoing reductions in antibiotic and zinc oxide use in farming. Plant host shift: The viability and persistence of a polyphagous, non-native insect Assessing the effects of a plant-host shift is important for monitoring insect populations over long time periods and for interventions in a conservation or pest management framework. In a heterogeneous environment, individuals may disperse between sources and sinks in order to persist. Here we propose a single-species two-patch model that aims to capture the generational movement of an insect that exhibits density-dependent dispersal, to see how shifting between hosts could alter its viability and asymptotic dynamics. We then analyse the stability and persistence properties of the model and further validate it using parameter estimates derived from laboratory experiments. In order to evaluate the potential of this model, we applied it to Drosophila suzukii (Diptera: Drosophilidae), which has become a harmful pest in several countries around the world. Although many studies have investigated the preference and attractiveness of potential hosts on this invasive drosophilid, no studies thus far have investigated whether a shift of fruit host could affect such a species ecological viability or spatiotemporal persistence. The model results show that a shift in host choice can significantly affect the growth potential and fecundity of a species such as D. suzukii, which ultimately could aid such invasive populations in their ability to persist within a changing environment. Bioinspired Dual-Functional Solid Lipid Nanoformulations for Targeted Drug Delivery and Sustained Release for Enhancement of Potency of Albendazole, an Antihelminthic Drug Targeted delivery has not been achieved for anthelmintic treatment, resulting in the requirement of excess drugs dose leading to side effects and therapeutic resistance. Gastrointestinal helminths ingest lipid droplets from digestive fluid for energy production, development, and defense. Worms habit inspired us to develop biocompatible, oral administrable, bee-wax derived solid lipid nanoparticles (SLN) with excellent drug (albendazole) loading efficiency of 83.3 {+/-} 6.5 mg/g and sustained-release properties (86.4 {+/-} 3.9 % of drug released within 24 h). Rhodamine B-loaded SLN showed time-dependent release and distribution of dye in vivo in Haemonchous contortus. The intestinal sustained-release property was shown by the particles that caused enhancement of albendazole potency for up to 50 folds. Therefore, this formulation has immense potential as an anthelminthic drug delivery vehicle that will not only be able to reduce the dose but will also reduce the drug-induced side effects by enhancing the bioavailability of the drug.

HighlightsO_LIAlbendazole-loaded Solid Lipid Nanoparticles (SLN-A) were formulated using Beeswax as stating material showing high drug loading capacity of 83 mg/g with sustained-release properties and 84 {+/-} 3 % of drug release within 24 h.
C_LIO_LISLN-A particles showed 50 fold enhancement of Albendazole activity against Haemonchous contortus worm.
C_LIO_LIRhodamine B-loaded SLN particles showed the specific uptake and in-vivo sustained release of dye in the worm.
C_LI 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. Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7 ko plants is accompanied by enhanced expression of carbohydrate transporters. Callose deposition around sieve pores, under control of callose synthase 7 (AtCALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were therefore employed to unveil the mode of involvement of CALS7 in the plants response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild-type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Author SummaryPhytoplasma infections are one of the most limiting factors for production of important crops all over the world. Phytoplasma disease epidemics can be handled mainly by insect-vector control using insecticides. Basic information about plant-phytoplasma interactions are still limited, nevertheless it is necessary to design new management and breeding strategies aimed to obtain more tolerant or resistant cultivars. Phytoplasmas are obligate intracellular parasites restricted to the phloem sieve tubes. Callose deposition at the sieve plates has been described since the  70s as a mechanical defence process to limit pathogen spread by occluding sieve pores. Studies reported 40 years later demonstrated that callose at the sieve pores is also involved in sieve-pore development and function and, hence, in mass-flow regulation, carbohydrate metabolism and distribution, and plant growth. Here, we reported on the role(s) of sieve-element callose in phytoplasma-infected Arabidopsis, using a mutant lacking AtCALS7, the enzyme responsible for callose synthesis in the sieve elements. The results indicate that loss of AtCAL7 appears to confer increased susceptibility to phytoplasma infection, due to alterations in expression of genes involved in sugar metabolism and membrane transport. In the long run, the identification of plant resistance or susceptibility traits against phytoplasmas will allow a complete re-organization of chemical control strategies, with obvious opportunities of reducing insecticide burden. A Hierarchy of Biomolecular Proportional-Integral-Derivative Feedback Controllers for Robust Perfect Adaptation and Dynamic Performance Proportional-Integral-Derivative (PID) feedback controllers have been the most widely used controllers in industry for almost a century due to their good performance, simplicity, and ease of tuning. Motivated by their success in various engineering disciplines, PID controllers recently found their way into synthetic biology, where the design of feedback molecular control systems has been identified as an important goal. In this paper, we consider the mathematical realization of PID controllers via biomolecular interactions. We propose an array of topologies that offer a compromise between simplicity and high performance. We first demonstrate that different Proportional-Integral (PI) controllers exhibit different capabilities for enhancing the dynamics and reducing variance (cell-to-cell variability). Next, we introduce several derivative controllers that are realized based on incoherent feedforward loops acting in a feedback configuration. Alternatively, we show that differentiators can be realized by placing molecular integrators in a negative feedback loop--an arrangement that can then be augmented by PI components to yield PID feedback controllers. We demonstrate that the derivative component can be exploited for enhancing system stability, dramatically increasing the molecular control systems dynamic performance, and for reducing the noise effect on the output of interest. The PID controller features are established through various deterministic and stochastic analyses as well as numerical simulations. Finally, we provide an experimental demonstration using a recently developed hybrid setup, the cyberloop, where the controller is implemented in silico to control a biological genetic circuit in vivo. The large array of novel biomolecular PID controllers introduced here forms a basis for the design and construction of advanced high-performance biomolecular control systems that robustly regulate the dynamics of living systems. Predicting gene regulatory networks from multi-omics to link genetic risk variants and neuroimmunology to Alzheimers disease phenotypes BackgroundGenome-wide association studies have found many genetic risk variants associated with Alzheimers disease (AD). However, how these risk variants affect deeper phenotypes such as disease progression and immune response remains elusive. Also, our understanding of cellular and molecular mechanisms from disease risk variants to various phenotypes is still limited. To address these problems, we performed an integrative multi-omics analysis of genotype, transcriptomics, and epigenomics for revealing gene regulatory mechanisms from disease variants to AD phenotypes.

MethodFirst, given the population gene expression data of a cohort, we construct and cluster its gene co-expression network to identify gene co-expression modules for various AD phenotypes. Next, we predict transcription factors (TFs) regulating co-expressed genes and AD risk SNPs that interrupt TF binding sites on regulatory elements. Finally, we construct a gene regulatory network (GRN) linking SNPs, interrupted TFs, and regulatory elements to target genes and gene modules for each phenotype in the cohort. This network thus provides systematic insights into gene regulatory mechanisms from risk variants to AD phenotypes.

ResultsOur analysis predicted GRNs in three major AD-relevant regions: Hippocampus, Dorsolateral Prefrontal Cortex (DLPFC), Lateral Temporal Lobe (LTL). Comparative analyses revealed cross-region-conserved and region-specific GRNs, in which many immunological genes are present. For instance, SNPs rs13404184 and rs61068452 disrupt SPI1 binding and regulation of AD gene INPP5D in the Hippocampus and LTL. However, SNP rs117863556 interrupts bindings of REST to regulate GAB2 in DLPFC only. Driven by emerging neuroinflammation in AD, we used Covid-19 as a proxy to identify possible regulatory mechanisms for neuroimmunology in AD. To this end, we looked at the GRN subnetworks relating to genes from shared AD-Covid pathways. From those subnetworks, our machine learning analysis prioritized the AD-Covid genes for predicting Covid-19 severity. Decision Curve Analysis also validated our AD-Covid genes outperform known Covid-19 genes for classifying severe Covid-19 patients. This suggests AD-Covid genes along with linked SNPs can be potential novel biomarkers for neuroimmunology in AD. Finally, our results are open-source available as a comprehensive functional genomic map for AD, providing a deeper mechanistic understanding of the interplay among multi-omics, brain regions, gene functions like neuroimmunology, and phenotypes. Characterization of methylation profiles in spontaneous preterm birth placental villous tissue Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing. Circulating immune complexes drive immunopathology in COVID-19 A dysregulated immune response with high levels of SARS-CoV-2 specific IgG antibodies characterizes patients with severe or critical COVID-19. Although a robust IgG response is traditionally considered to be protective, excessive triggering of activating Fc-gamma-receptors (Fc{gamma}Rs) could be detrimental and cause immunopathology. Here, we document that patients who develop soluble circulating IgG immune complexes (sICs) during infection are subject to enhanced immunopathology driven by Fc{gamma}R activation. Utilizing cell-based reporter systems we provide evidence that sICs are predominantly formed prior to a specific humoral response against SARS-CoV-2. sIC formation, together with increased afucosylation of SARS-CoV-2 specific IgG eventually leads to an enhanced CD16 (Fc{gamma}RIII) activation of immune cells reaching activation levels comparable active systemic lupus erythematosus (SLE) disease. Our data suggest a vicious cycle of escalating immunopathology driven by an early formation of sICs in predisposed patients. These findings reconcile the seemingly paradoxical findings of high antiviral IgG responses and systemic immune dysregulation in severe COVID-19.

Clinical implicationsThe identification of sICs as drivers of an escalating immunopathology in predisposed patients opens new avenues regarding intervention strategies to alleviate critical COVID-19 progression.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/449893v4_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@b4616corg.highwire.dtl.DTLVardef@682d1aorg.highwire.dtl.DTLVardef@16946cborg.highwire.dtl.DTLVardef@a6ef7d_HPS_FORMAT_FIGEXP  M_FIG C_FIG A vicious cycle of immunopathology in COVID-19 patients is driven by soluble multimeric immune complexes (sICs). SARS-CoV-2 infection triggers sIC formation in prone individuals. Activation of Fc{gamma}RIII/CD16 expressing immune cells by sICs precedes a humoral response to SARS-CoV2 infection. sICs and infection add to IgG afucosylation, further enhancing Fc{gamma}RIII/CD16 activation by opsonized targets. High inflammation induces further sIC mediated immune cell activation ultimately leading to an escalating immunopathology. High performance single-cell gene regulatory network inference atscale: The Inferelator 3.0 MotivationGene regulatory networks define regulatory relationships between transcription factors and target genes within a biological system, and reconstructing them is essential for understanding cellular growth and function. Methods for inferring and reconstructing networks from genomics data have evolved rapidly over the last decade in response to advances in sequencing technology and machine learning. The scale of data collection has increased dramatically; the largest genome-wide gene expression datasets have grown from thousands of measurements to millions of single cells, and new technologies are on the horizon to increase to tens of millions of cells and above.

ResultsIn this work, we present the Inferelator 3.0, which has been significantly updated to integrate data from distinct cell types to learn context-specific regulatory networks and aggregate them into a shared regulatory network, while retaining the functionality of the previous versions. The Inferelator is able to integrate the largest single-cell datasets and learn cell-type specific gene regulatory networks. Compared to other network inference methods, the Inferelator learns new and informative Saccharomyces cerevisiae networks from single-cell gene expression data, measured by recovery of a known gold standard. We demonstrate its scaling capabilities by learning networks for multiple distinct neuronal and glial cell types in the developing Mus musculus brain at E18 from a large (1.3 million) single-cell gene expression dataset with paired single-cell chromatin accessibility data.

AvailabilityThe inferelator software is available on GitHub (https://github.com/flatironinstitute/inferelator) under the MIT license and has been released as python packages with associated documentation (https://inferelator.readthedocs.io/). Hidden dynamics of proteasome autoregulation discovered by cryo-EM data-driven deep learning The cellular functions are executed by macromolecular complexes in nonequilibrium dynamic processes, which exhibit a vast diversity of conformational states. Solving conformational continuum of important complexes at atomic level remains a key challenge in the methodology of computational biophysics. Here we introduce a deep-learning framework, named AlphaCryo4D, which enables atomic-level cryogenic electron microscopy reconstructions of nonequilibrium conformational continuum. AlphaCryo4D integrates 3D deep residual learning with manifold embedding of energy landscapes, which simultaneously improves 3D classification accuracy and reconstruction resolution via an energy-based particle-voting algorithm. Applications of this methodology to six experimental datasets demonstrated its generality in breaking resolution barrier of visualizing dynamic components of functional complexes, in choreographing continuous inter-subunit motions and in exploring their  hidden conformational space. Importantly, AlphaCryo4D identified 64 conformers of the substrate-bound human 26S proteasome and reconstituted single-nucleotide-exchange dynamics of proteasomal AAA-ATPase motor during translocation initiation, which together illuminates a grand hierarchical allostery for proteasome autoregulation. A dopamine-dependent decrease in dorsomedial striatum direct pathway neuronal activity is required for learned motor coordination It has been suggested that the dorsomedial striatum (DMS) facilitates the early stages of motor learning for goal-directed actions, whereas at later stages, control is transferred to the dorsolateral striatum (DLS), which enables the motor actions to become a skill or habit. It is unknown whether these striatal regions are simultaneously active while expertise is acquired during skill learning. To address this question, we developed the "treadmill training task" to track changes in mouse locomotor coordination during practice at running that simultaneously provides a means to measure local neuronal activity using photometry. We analyzed body position and paw movement to evaluate changes in motor coordination over practice sessions on the treadmill using DeepLabCut and custom-built code. By correlating improvements in motor coordination during training with simultaneous neuronal calcium activity in the striatum, we found that DMS direct pathway neurons exhibited decreased activity as the mouse gains proficiency at running. In contrast, direct pathway activity in the DLS was similar throughout training and did not correlate with learned skill proficiency. Pharmacological blockade of D1 dopamine receptors in these subregions during task performance confirmed that dopamine neurotransmission in the DMS direct pathway activity is necessary for efficient motor coordination learning, while dopamine signalling in the DLS is important for both coordination learning and maintenance of the acquired skill. These results provide new tools to measure changes in fine motor skills during simultaneous recordings of brain activity, revealing fundamental features of the neuronal substrates of motor learning. Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level 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. R7 photoreceptor axon targeting depends on the relative levels of lost and found expression in R7 and its synaptic partners As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections. Coordination of two opposite flagella allows high-speed swimming and active turning of individual zoospores Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when these biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, characteristics of individual swimming of zoospores have not been fully investigated. It remains unknown about the characteristics of two opposite beating flagella during translation and turning, and the roles of each flagellum on zoospore swimming. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that turning involves a complex active process, in which the posterior flagellum temporarily stops, while the anterior flagellum keeps on beating and changes its pattern from sinusoidal waves to power and recovery strokes, similar to Chlamydomonass breaststroke, to reorient its body to a new direction. Our study is a fundamental step towards a better understanding of the spreading of plant pathogens motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated "run-and-tumble" motility class exhibited by peritrichous bacteria. A cell-based screen identifies HDAC inhibitors as activators of RIG-I signaling Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Combination treatment of tumor cell lines with Entinostat increased RIG-I induced cell death in a mammary carcinoma cell line that is resistant to either Entinostat or RIG-I agonist alone. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. GUIdEStaR (G-quadruplex, uORF, IRES, Epigenetics, Small RNA, Repeats), the integrated metadatabase in conjunction with neural network methods GUIdEStaR integrates existing databases of various types of G-quadruplex, upstream Open Reading Frame (uORF), Internal Ribosome Entry Site (IRES), methylation to RNA and histone protein, small RNA, and repeats. GUIdEStaR consists of approx. 40,000 genes and 320,000 transcripts. An mRNA transcript is divided into 5 regions (5UTR, 3UTR, exon, intron, and biological region) where each region contains presence-absence data of 169 different types of elements. Recently, artificial intelligence (AI) based analysis of sequencing data has been gaining popularity in the area of bioinformatics. GUIdEStaR generates datasets that can be used as inputs to AI methods. At the GUIdEStaR homepage, users submit gene symbols by clicking a "Send" button, and shortly result files in CSV format are available for download at the result website. Users have an option to send the result files to their email addresses. Additionally, the entire database and the example Java codes are also freely available for download. Here, we demonstrate the database usage with three neural network classification studies-1) small RNA study for classifying transcription factor (TF) genes into either one of TF mediated by small RNA originated from SARS-CoV-2 or by human microRNA (miRNA), 2) cell membrane receptor study for classifying receptor genes as either with virus interaction or without one, and 3) nonsense mediated mRNA decay (NMD) study for classifying cell membrane and nuclear receptors as either NMD target or non-target. GUIdEStaR is available for access to the easy-to-use web-based database at www.guidestar.kr and for download at https://sourceforge.net/projects/guidestar. The Consequences of Differential Origin Licensing Dynamics in Distinct Chromatin Environments MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability. IRI-CCE: A Platform for Evaluating CRISPR-based Base Editing Tools and Its Components Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs. 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. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. The pH Dependence of Niclosamide Solubility, Dissolution, and Morphology Motivates Potentially Universal Mucin-Penetrating Nasal and Throat Sprays for COVID19, its Contagious Variants, and Other Respiratory Viral Infections MotivationWith the coronavirus pandemic still raging, prophylactic nasal and early treatment throat sprays could help prevent infection and reduce viral load. Niclosamide has the potential to treat a broad range of viral infections if local bioavailability is optimized as mucin-penetrating solutions as opposed to more traditional microparticle-based sprays that cannot penetrate the mucin.

ExperimentalpH-dependence of supernatant concentrations and dissolution rates of niclosamide were measured in buffered solutions by Nanodrop-UV/Vis-spectroscopy for niclosamide from different suppliers, as precipitated material, and as cosolvates. Data was compared to predictions from Henderson Hasselbalch and precipitation pH models. Optimal microscopy was used to observe the morphologies of precipitated and converted niclosamide.

ResultsSupernatant-concentrations of niclosamide increased with increasing pH, from 1.77uM at pH 3.66 to 30uM at pH 8, and more rapidly from 90uM at pH8.5 to 300uM at pH 9.1, reaching 641uM at pH 9.5. Logarithmic rates for dissolution increased by [~]3x for pHs 8.62 to 9.44. However, when precipitated from supersaturated solution, niclosamide equilibrated to much lower final supernatant concentrations, reflective of more stable polymorphs at each pH that were also apparent for niclosamide from other suppliers and cosolvates.

ConclusionsGiven niclosamides activity against COVID19, its more contagious variants, and other respiratory viral infections, these niclosamide solutions, that put the virus in lockdown, could represent universal prophylactic nasal and early treatment throat sprays. As solutions they would be the simplest and potentially most effective formulations from both an efficacy standpoint as well as manufacturing and distribution, with no cold chain. They now just need testing. Mate sharing in male stump-tailed macaques as a possible case of coalition-like behavior to modify the group-wise fitness distribution In multi-male multi-female groups of animals, male reproductive success is often skewed toward top-ranking males. Coalition formation by middle- to low-rankers can be seen as a collaborative effort to modify the distribution of reproductive success within the group, so that they gain more than they would do otherwise. It has been pointed out, on the other hand, that a coalition of top-ranking males could hardly be profitable in the sense that they would gain little additional benefit from making collaborative effort. Here we report our novel observation of facultative sharing of mating opportunities among males in a wild population of stump-tailed macaques (Macaca arctoides) as a possible case of coalition-like behavior in which dominant males jointly guard females from mating with subordinate males and actively share mating opportunities within the allies. First, we report our novel observation of facultative sharing of mating opportunities in male stump-tailed macaques, where two or more males remain in close proximity to and copulate with a female in turn without contesting or sneaking. Second, considering the kind of coalition formation in which dominant males collaboratively exclude subordinates from mating competition and thereby strengthen the reproductive skew that already exists, we specify, by means of mathematical modeling, the condition for this kind of coalition formation to be selectively favored. Finally, we derive predictions about the occurrence of the coalition-like behavior depending on ecological and demographic factors, and test them empirically using data from the five groups of stump-tailed macaques in our study population.