Acetyltransferases GCN5 and PCAF are required for B lymphocyte maturation in mice B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an open chromatin state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacks both GCN5 and PCAF in B cells. We found that double-deficient mice possess low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We conclude that both GCN5 and PCAF are required for B cell development in vivo.
Immune complex solubility and size govern Fc-gamma receptor responses Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings.
14-3-3ζ constrains insulin secretion by regulating mitochondrial function in pancreatic β-cells While critical for neurotransmitter synthesis in the brain, members of the 14-3-3 protein family are often assumed to have redundant, over-lapping roles due to their high sequence homology and ubiquitous expression. Despite this assumption, various mammalian 14-3-3 isoforms have now been implicated in regulating cellular and organismal metabolism; however, these functions were primarily observed in cell lines or from systemic knockout mouse models. To date, we have begun to define the contributions of 14-3-3{zeta} in adipocytes, but whether 14-3-3{zeta} has additional metabolic roles in other cell types, such as the pancreatic {beta}-cell, is unclear. We previously documented a pro-survival role of 14-3-3{zeta} in MIN6 insulinoma cells, as depletion of 14-3-3{zeta} induced cell death, but paradoxically, whole-body deletion of 14-3-3{zeta} in mice resulted in significantly enlarged {beta}-cell area with no effects on insulin secretion. To better understand the role of 14-3-3{zeta} in {beta}-cells, we generated {beta}-cell-specific 14-3-3{zeta} knockout ({beta}14-3-3{zeta}KO) mice, and while no differences in {beta}-cell mass were observed, {beta}14-3-3{zeta}KO mice displayed potentiated insulin secretion due to enhanced mitochondrial function and ATP synthesis. Deletion of 14-3-3{zeta} led to profound changes to the {beta}-cell transcriptome, where pathways associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute treatment of mouse islets and human islets with pan-14-3-3 inhibitors recapitulated the potentiation in glucose-stimulated insulin secretion (GSIS) and mitochondrial function, suggesting that 14-3-3{zeta} is a critical isoform in {beta}-cells that regulates GSIS. In dysfunctional db/db islets and islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3{zeta}, was inversely associated with insulin secretory capacity, and pan-14-3-3 protein inhibition was capable of enhancing GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3{zeta} and its related isoforms in insulin secretion and mitochondrial function in {beta}-cells. A deeper understanding of how 14-3-3{zeta} influences {beta}-cell function will further advance our knowledge of how insulin secretion from {beta}-cells is regulated. 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. 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. Induction of exaggerated cytokine production in human peripheral blood mononuclear cells by a recombinant SARS-CoV-2 spike glycoprotein S1 and its inhibition by dexamethasone An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNF, IL-6, IL-1{beta} and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-{kappa}B p65 and I{kappa}B, and I{kappa}B degradation. DNA binding of NF-{kappa}B p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 {micro}M) resulted in inhibition of spike glycoprotein S1-induced NF-{kappa}B activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNF, IL-6, IL-1{beta} and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1{beta} production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-{kappa}B, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 is possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm. Feed efficiency of lactating Holstein cows was not as repeatable across diets as within diet over subsequent lactation stages BackgroundImproving feed efficiency has become a common target for dairy farmers to meet the requirement of producing more milk with fewer resources. To improve feed efficiency, a prerequisite is to ensure that the cows identified as most or least efficient will remain as such, independently of diet composition. Therefore, the current research analysed the ability of lactating dairy cows to maintain their feed efficiency while changing the energy density of the diet by changing its concentration in starch and fibre. A total of 60 lactating Holstein cows, including 33 primiparous cows, were first fed a high starch diet-low fibre (diet S+F-), then switched over to a low starch diet-high fibre (diet S-F+). To know if diet affect feed efficiency, we compared the ability of feed efficiency to be maintained within a diet over subsequent lactation stages, known as repeatability of feed efficiency, with its ability to be maintained across diets, known as reproducibility of feed efficiency. To do so we used two indicators: the estimation of the error of repeatability/reproducibility, which is commonly used in metrology, and the coefficient of correlation of concordance (CCC), which is used in biology. The effect of diet change could also lead to a change in cows sorting behaviour which could potentially affect feed efficiency if for example the most efficient cows select more concentrate than the least efficient. We therefore analysed the relationship between the differences in individual feed refusals composition and the differences in feed efficiency. To do so, the composition of each feed refusal was described with its near infra-red (NIR) spectroscopy and was performed on each individual feed ingredient, diet and refusals and used as composition variable. The variability of the NIR spectra of the refusals was described with its principal components thanks to a principal component analysis (PCA). The Pearson correlation was estimated to check the relationship between feed efficiency and refusals composition, i.e. sorting behaviour.

ResultsThe error of reproducibility of feed efficiency across diets was 2.95 MJ/d. This error was significantly larger than the errors of repeatability estimated within diet, which were 2.01 MJ/d within diet S-F+ and 2.40 MJ/d within diet S+F-. The CCC was 0.64 between feed efficiency estimated within diet S+F- and feed efficiency estimated within diet S-F+. This CCC was smaller than the one observed for feed efficiency estimated within diet between two subsequent lactation stages (CCC = 0.72 within diet S+F- and 0.85 within diet S-F+). Feed efficiency was poorly correlated to the first two principal components, which explained 90% of the total variability of the NIR spectra of the individual refusals. This suggests that feed sorting behaviour did not explain differences in feed efficiency.

ConclusionsFeed efficiency was significantly less reproducible across diets than repeatable within the same diet over subsequent lactation stages, but cows ranking for feed efficiency was not significantly affected by diet change. This loss in repeatability across diets could be due to a more pronounced feed sorting subsequent to the change in diet composition. However, the differences in sorting behaviour between cows were not associated to feed efficiency differences in this trial neither with the S+F- diet nor with the S-F+ diet. Those results have to be confirmed on diets having different forage to concentrate ratios to ensure that the least and most efficient cows will not change. Defining An Expanded RAS Conformational Landscape Based on Over 700 Experimentally Determined Structures of KRAS, NRAS, and HRAS For many human cancers and tumor-associated diseases, mutations in the RAS isoforms (KRAS, NRAS, and HRAS) are the most common oncogenic alterations, making these proteins high-priority therapeutic targets. Effectively targeting the RAS isoforms requires an exact understanding of their active, inactive, and druggable conformations. However, there is no structure-guided catalogue of RAS conformations to guide therapeutic targeting or examining the structural impact of RAS mutations. We present an expanded classification of RAS conformations based on analyzing their catalytic switch 1 (SW1) and switch 2 (SW2) loops. From all 721 available human KRAS, NRAS, and HRAS structures in the Protein Data Bank (PDB) (206 RAS-protein complexes, 190 inhibitor-bound, and 325 unbound, including 204 WT and 517 mutated structures), we created a broad conformational classification based on the spatial positions of residue Y32 in SW1 and residue Y71 in SW2. Subsequently, we defined additional conformational subsets (some previously undescribed) by clustering all well modeled SW1 and SW2 loops using a density-based machine learning algorithm with a backbone dihedral-based distance metric. In all, we identified three SW1 conformations and nine SW2 conformations, each which are associated with different nucleotide states (GTP-bound, nucleotide-free, and GDP-bound) and specific bound proteins or inhibitor sites. The GTP-bound SW1 conformation can be further subdivided based on the hydrogen (H)-bond type made between residue Y32 and the GTP {gamma}-phosphate: water-mediated, direct, or no H-bond. Further analyzing these structures clarified the catalytic impact of the G12D and G12V RAS mutations, and the inhibitor chemistries that bind to each druggable RAS conformation. To facilitate future RAS structural analyses, we have created a web database, called Rascore, presenting an updated and searchable dataset of human KRAS, NRAS, and HRAS structures in the PDB, and which includes a page for analyzing user uploaded RAS structures by our algorithm (http://dunbrack.fccc.edu/rascore/).

SignificanceAnalyzing >700 experimentally determined RAS structures helped define an expanded landscape of active, inactive and druggable RAS conformations, the structural impact of common RAS mutations, and previously uncharacterized RAS-inhibitor binding modes. Trade-offs in biosensor optimization for dynamic pathway engineering Recent progress in synthetic biology allows the construction of dynamic control circuits for metabolic engineering. This technology promises to overcome many challenges encountered in traditional pathway engineering, thanks to their ability to self-regulate gene expression in response to bioreactor perturbations. The central components in these control circuits are metabolite biosensors that read out pathway signals and actuate enzyme expression. However, the construction of metabolite biosensors is a major bottleneck for strain design, and a key challenge is to understand the relation between biosensor dose-response curves and pathway performance. Here we employ multiobjective optimization to quantify performance trade-offs that arise in the design and calibration of metabolite biosensors. Our approach reveals strategies for tuning dose-response curves along an optimal trade-off between production flux and the cost of an increased expression burden on the host. We explore properties of control architectures built in the literature, and identify their advantages and caveats in terms of performance and robustness to growth conditions and leaky promoters. We demonstrate the optimality of a control circuit for glucaric acid production in Escherichia coli, which has been shown to increase titer by 2.5-fold as compared to static designs. Our results lay the groundwork for the automated design of control circuits for pathway engineering, with applications in the food, energy and pharmaceutical sectors. Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-{kappa}B from DNA is achieved through accelerated dissociation by the inhibitor protein I{kappa}B. Using single-molecule FRET, we observe a continuum of conformations of NF-{kappa}B in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-{kappa}B is partially bound, allowing I{kappa}B invasion to facilitate DNA dissociation. I{kappa}B induces a locked conformation where the DNA-binding domains of NF-{kappa}B are too far apart to bind DNA, whereas the loss-of-function I{kappa}B mutant retains the NF-{kappa}B conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors. MODEL PREDICTIVE CONTROL OF CANCER CELLULAR DYNAMICS: A NEW STRATEGY FOR THERAPY DESIGN Recent advancements in Cybergenetics have led to the development of new computational and experimental platforms that enable to robustly steer cellular dynamics by applying external feedback control. Such technologies have never been applied to regulate intracellular dynamics of cancer cells. Here, we show in silico that adaptive model predictive control (MPC) can effectively be used to steer signalling dynamics in Non-Small Cell Lung Cancer (NSCLC) cells to resemble those of wild-type cells, and to support the design of combination therapies. Our optimisation-based control algorithm enables tailoring the cost function to force the controller to alternate different drugs and/or reduce drug exposure, minimising both drug-induced toxicity and resistance to treatment. Our results pave the way for new cybergenetics experiments in cancer cells for drug combination therapy design. Adolescent Alcohol Binge-Drinking induces delayed appearance of behavioral defects in mice Adolescence is a developmental period characterized by significant changes in brain architecture and behavior. The immaturity of the adolescent brain is associated with heightened vulnerability to exogenous agents, including alcohol. Alcohol is the most consumed drug among teenagers, and binge-drinking during adolescence is a major public health concern. Studies have suggested that adolescent alcohol exposure (AAE) may interfere with the maturation of frontal brain regions and lead to long-lasting behavioral consequences. In this study, we used a mouse model of AAE in which adolescent mice reach high blood alcohol concentration after voluntary binge-drinking. In order to assess short- and long-term consequences of AAE, a battery of behavioral tests was performed during late adolescence and during adulthood. We showed that AAE had no short-term effect on young mice behavior but rather increased anxiety- and depressive-like behaviors, as well as alcohol consumption during adulthood. Moreover, alcohol binge-drinking during adolescence dramatically decreased recognition memory performances and behavioral flexibility in both adult males and females. Furthermore, we showed that voluntary consumption of alcohol during adolescence did not trigger any major activation of the innate immune system in the prefrontal cortex (PFC). Together, our data suggest that voluntary alcohol binge-drinking in adolescent mice induces a delayed appearance of behavioral impairments in adulthood. Genetic coupling of life-history and aerobic performance in Atlantic salmon indicates potential for coevolution across life-stages A better understanding of the genetic and phenotypic architecture underlying life-history variation is a longstanding aim in biology. Theories suggest energy metabolism determines life-history variation by modulating resource acquisition and allocation trade-offs, but the genetic underpinnings of the relationship and its dependence on ecological conditions have rarely been demonstrated. The strong genetic determination of age-at-maturity by two unlinked genomic regions (vgll3 and six6) makes Atlantic salmon (Salmo salar) an ideal model to address these questions. Using more than 250 juveniles in common garden conditions, we quantified the covariation between metabolic phenotypes -standard and maximum metabolic rates (SMR and MMR), and aerobic scope (AS) - and the life-history genomic regions and tested if food availability modulates the relationships. We found that the early maturation genotype in vgll3 was associated with higher MMR and consequently AS. Additionally, MMR exhibited physiological epistasis; it was decreased when late maturation genotypes co-occurred in both genomic regions. Contrary to our expectation, the life-history genotypes had no effects on SMR. Further, food availability had no effect on the genetic covariation, suggesting a lack of genotype-by-environment interactions. Our results provide insights on the key organismal processes that link energy use at the juvenile-stage to age-at-maturity, indicating potential mechanisms by which metabolism and life-history can coevolve. Modeling miRNA-driven post-transcriptional regulatory signals by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses. A rationalized definition of general tumor suppressor microRNAs excludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors. Potential geographic distribution and ecological niche of New World dobsonflies (Megaloptera, Corydalidae): the case of the Neartic-Neotropical transition zone The dobsonflies (Megaloptera, Corydalidae) are an interesting group of insects, but among the New World dobsonflies, it is not known the effect of the Neartic-Neotropical transition zone on their biogeographic distribution. Here we studied at the species level, the records on the geographic range of the dobsonflies that occurred in and near the transition zone. We presented potential geographic distributions based on ecological niche models for several species of dobsonflies. Results suggested that the geographic range of dobsonflies in the transition zone is associated to mountainous formations and that most species favor warm climates with higher precipitation rates. Climate types tend to be important for species that show narrow geographic ranges, but precipitation tends to be the most important variable to explain species dispersion. Overall, our models support the dispersion of dobsonflies from the Neotropics to North America and explain the two endemic species in Mexico as the result of the formation of the transition zone. Hydrophobic mismatch is a key factor in protein transport on the Tat pathway The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoid, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits, TatA, TatB and TatC. TatA and TatB from all bacterial species possess short transmembrane alpha-helices (TMHs), both of which are only fifteen residues long in E. coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer. Here, by modifying the length of the TMHs of E. coli TatA and TatB, we assess the functional importance of the hydrophobic mismatch in the Tat transport mechanism. Surprisingly, both TatA and TatB with as few as 11 residues in their respective TMHs are still able to insert into the membrane bilayer, albeit with a decline in membrane integrity. Three different assays, both qualitative and quantitative, were conducted to evaluate the Tat activity of the TMH length mutants. Our experiments indicate that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off with any modification of this length. We believe our study supports a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation, and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes. Cytosolic EZH2-IMPDH2 complexes regulate melanoma progression via GTP The enhancer of zeste homolog 2 (EZH2) oncoprotein is a histone methyltransferase that functions canonically as a catalytic subunit of the polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 at Lys 27 (H3K27me3). Although targeting EZH2 methyltransferase is a promising therapeutic strategy against cancer, methyltransferase-independent oncogenic functions of EZH2 are described. Moreover, pharmacological EZH2 methyltransferase inhibition was only variably effective in pre-clinical and clinical studies, suggesting that targeting EZH2 methyltransferase alone may be insufficient. Here, we demonstrate a non-canonical mechanism of EZH2s oncogenic activity characterized by interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and downstream promotion of guanosine-5-triphosphate (GTP) production. EZH2-IMPDH2 interactions identified by Liquid Chromatography-Mass Spectrometry (LC-MS) of EZH2 immunoprecipitates from melanoma cells were verified to occur between the N-terminal EED-binding domain of cytosolic EZH2 and the CBS domain of IMPDH2 in a methyltransfersase-independent manner. EZH2 silencing reduced cellular GTP, ribosome biogenesis, RhoA-mediated actomyosin contractility and melanoma cell proliferation and invasion by impeding the activity of IMPDH2. Guanosine, which replenishes GTP, reversed these effects and thereby promoted invasive and clonogenic cell states even in EZH2 silenced cells. IMPDH2 silencing antagonized the proliferative and invasive effects of EZH2, also in a guanosine-reversible manner. In human melanomas, high cytosolic EZH2 and IMPDH2 expression were associated with nucleolar enlargement, a marker of ribosome biogenesis. EZH2-IMPDH2 complexes were also observed in a range of cancers in which Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic, although notably non-toxic in normal cells. These findings illuminate a previously unrecognized, non-canonical, methyltransferase-independent, and GTP-dependent mechanism by which EZH2 regulates tumorigenicity in melanoma and other cancers, opening new avenues for development of anti-EZH2 therapeutics.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/467024v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@153dd35org.highwire.dtl.DTLVardef@dfeb43org.highwire.dtl.DTLVardef@ce8148org.highwire.dtl.DTLVardef@189d8da_HPS_FORMAT_FIGEXP  M_FIG Graphical Abstract C_FIG HighlightsO_LIEZH2 has non-canonical methyltransferase-independent and GTP-dependent tumorigenic and metastatic functions in melanoma.
C_LIO_LIThe N-terminal EED-binding domain of EZH2 interacts with the CBS domain of IMPDH2 in a polycomb repressive complex 2- (PRC2-) and methylation-independent manner.
C_LIO_LIEZH2 accumulates with IMPDH2 in the cytoplasm and increases IMPDH2s tetramerization-mediated activity independently of EZH2 methyltransferase.
C_LIO_LIEZH2 upregulates GTP synthesis by IMPDH2 activation and thereby activates ribosome biogenesis via rRNA synthesis and actomyosin contractility via RhoA GTPase.
C_LIO_LISappanone A (SA) inhibits IMPDH2-EZH2 interactions and is anti-proliferative across a range of cancers including melanoma, but not in normal cells.
C_LI 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. Analysis of caspase-mediated suppression of the cGAS/STING pathway in Kaposi's sarcoma associated herpesvirus lytic infection reveals a dramatic cellular heterogeneity in type I interferon responses As a result of the ongoing virus-host arms race, viruses have evolved numerous immune subversion strategies, many of which are aimed at suppressing the production of type I interferons (IFNs)1. This focus on IFN evasion highlights the essential role of these cytokines in controlling viral infections. Apoptotic caspases have recently emerged as important regulators of type I IFN signaling in both non-infectious contexts and during viral infection2-5. Despite being widely considered anti-viral factors since they can trigger cell death, several apoptotic caspases promote viral replication by suppressing innate immune responses2-5. Indeed, we previously discovered that the AIDS-associated oncogenic gammaherpesvirus Kaposis sarcoma-associated herpesvirus (KSHV) exploits caspase-8 activity to suppress the antiviral type I IFN response and promote viral replication5. However, the mechanism of this novel viral immune evasion strategy is poorly understood, particularly how caspase-8 antagonizes IFN signaling during KSHV infection. Here we show that caspase activity inhibits the DNA sensor cGAS6 during KSHV lytic replication to block type I IFN induction. Furthermore, we use single-cell RNA-sequencing to reveal that the potent antiviral state conferred upon caspase inhibition is mediated by an exceptionally small percentage of IFN-{beta}-producing cells, thus uncovering further complexity of IFN regulation during viral infection. Collectively, these results provide insight into multiple levels of cellular type I IFN regulation that viruses co-opt for immune evasion. Unraveling these mechanisms can inform targeted therapeutic strategies for viral infections and reveal cellular mechanisms of regulating interferon signaling in the context of cancer and chronic inflammatory diseases. Strong immobilization of C. elegans en masse on cultivation plates by thermoelectric cooling for high-throughput submicron-resolution microscopy Despite its profound impact on biology, most high-resolution in vivo microscopy approaches remain low throughput because current immobilization techniques require significant manual effort. We greatly accelerate imaging of the nematode Caenorhabditis elegans by implementing a simple cooling approach to easily immobilize entire populations directly on their cultivation plates. We optimize and characterize cooling immobilization. Counterintuitively, relatively warmer temperatures immobilize animals significantly more effectively than colder temperatures utilized in prior studies. This enhanced immobilization enables clear submicron-resolution fluorescence imaging, which is challenging to achieve with most current immobilization techniques. We demonstrate 64x magnification 3D imaging and timelapse recording of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. By obviating individual animal manipulation, our approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows. Classification of daily crop phenology in PhenoCams using deep learning and hidden markov models Near surface cameras, such as those in the PhenoCam network, are a common source of ground truth data in modelling and remote sensing studies. Despite having locations across numerous agricultural sites, few studies have used near surface cameras to track the unique phenology of croplands. Due to management activities, crops do not have a natural vegetation cycle which many phenological extraction methods are based on. For example, a field may experience abrupt changes due to harvesting and tillage throughout the year. A single camera can also record several different plants due to crop rotations, fallow fields, and cover crops. Current methods to estimate phenology metrics from image time series compress all image information into a relative greenness metric, which discards a large amount of contextual information. This can include the type of crop present, whether snow or water is present on the field, the crop phenology, or whether a field lacking green plants consists of bare soil, fully senesced plants, or plant residue. Here we developed a modelling workflow to create a daily time series of crop type and phenology, while also accounting for other factors such as obstructed images and snow covered fields. We used a mainstream deep learning image classification model, VGG16. Deep learning classification models do not have a temporal component, so to account for temporal correlation among images our workflow incorporates a hidden markov model in the post-processing. The initial image classification model had out of sample F1 scores of 0.83-0.85, which improved to 0.86-0.91 after all post-processing steps. The resulting time series show the progression of crops from emergence to harvest, and can serve as a daily, local scale dataset of field states and phenological stages for agricultural research. Harnessing changes in open chromatin determined by ATAC-seq to generate insulin-responsive reporter constructs. BackgroundGene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed.

ResultsWe used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a strong correlation between chromatin regions annotated to distal promoters (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate distal promoter regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters.

ConclusionsInsulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression. Inflammatory response in hematopoietic stem and progenitor cells triggered by activating SHP2 mutations evokes blood defects Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with Shp2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies and in vivo transplantability of HSPCs. Single cell mRNA sequencing of HSPCs from Shp2D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies. GWAS-associated variants, non-genetic factors, and transient transcriptome in Multiple Sclerosis etiopathogenesis: a colocalization analysis A clinically actionable understanding of multiple sclerosis (MS) etiology goes through GWAS interpretation, prompting research on new gene regulatory models. We previously suggested a stochastic etiologic model where small-scale random perturbations could reach a threshold for MS development. The recently described mapping of the transient transcriptome (TT), including intergenic and intronic RNAs, seems appropriate to verify this model through a rigorous colocalization analysis. We show that genomic regions coding for the TT were significantly enriched for MS-associated GWAS variants and DNA binding sites for molecular transducers mediating putative, non-genetic, etiopathogenetic factors for MS (e.g., vitamin D deficiency, Epstein Barr virus latent infection, B cell dysfunction). These results suggest a model whereby TT-coding regions are hotspots of convergence between genetic ad non-genetic factors of risk/protection for MS, and plausibly for other complex disorders. Our colocalization analysis also provides a freely available data resource (www.mscoloc.com) for future research on MS transcriptional regulation. Molecular diagnosis of new isolate of tomato yellow leaf curl virus (TYLCV) in Iraq Tomato yield and quality in Iraq have been threatened by a variable range of infections caused by tomato yellow leaf curl virus. In previous studies, the TYLCV isolates were partially characterized using molecular tests for small fragments not the entire length of the virus. Sample of TYLCV-infected tomato has applied in this study to diagnose complete sequence of TYLCV isolate. Three sets of primers that belong to three well-identified strains in Iraq were used in a PCR technique and interestingly the results were negative. A new Iraqi isolate has been characterized as a first novel Iraqi isolate detected ever using next generation (NGS) and bioinformatics techniques. The NGS platform has produced about 78,232,062 paired reads of the TYLCV-infected tomato var. Oula F1. The complete raw reads of the infected variety have been analyzed using RepeatExplorer pipeline and Map to reference. The full sequence of TYLCV was reconstructed and extracted to consist of 2770 nt and then deposited in Genbank under accession number MT583814. Copy numbers and genome proportion of this sequence have been calculated that were 3523 and 0,086% respectively. Phylogenetic analyses of full nucleotide sequences confirmed close relationship to Iranian isolate (TYLCV-Kahnooj) than other published viruses. Additionally, a and {beta} DNA satellites have not discovered in the TYLCV-infected sample.