Inhibitory proteins block substrate access by occupying the active site cleft of Bacillus subtilis intramembrane metalloprotease SpoIVFB Intramembrane proteases function in numerous signaling pathways that impact health, but knowledge about regulation of intramembrane proteolysis is limited. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that BofA residues in and near a predicted transmembrane segment are required for SpoIVFB inhibition, and cross-linking experiments indicated that this transmembrane segment occupies the SpoIVFB active site cleft. SpoIVFA is also required for SpoIVFB inhibition. The inhibitory proteins block access of the substrate N-terminal Proregion to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a transmembrane segment and a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of intramembrane protease inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics.
Cell Intrinsic Control of Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells by pH Many biological processes are controlled by cell death and proliferation. The present study found that intracellular pH positively correlated with proliferation but negatively with apoptosis. Acid treatments lowered intracellular pH and preferentially depleted highly proliferating lymphocytes, whereas alkaline treatments increased intracellular pH and enhanced lymphocyte proliferation. Likewise, acid treatments induced apoptosis and alkaline treatments maintained proliferation of tumor cells. These findings support a unified theory of regulation of cell death and proliferation where an intracellular pH balance controls both events, and the mitochondria act as the pH-stats of the cell. Accordingly, the Warburg effect is explained as necessary for proliferating cells to have a high intracellular pH environment to accelerate proliferation and avoid low pH-induced death.
Blocking antibodies against integrin-a3, integrin-aM, and integrin-aMb2 de-differentiate myofibroblasts and reverse lung and kidney fibroses in a mouse model Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of the disease. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins 3, M, and M{beta}2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and reverse lung and kidney fibrosis in vivo. Decorins collagen-binding peptide directs blocking antibodies (against integrins-3, -M, -M{beta}2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to reverse fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment and reversal of fibrosis.

SummaryBlocking antibodies against integrins-3, -M, and -M{beta}2 can be targeted to sites of fibrosis, reverse lung and kidney fibroses, and offer the potential to bring immunotherapy to fibrosis Precartilage condensation during limb skeletogenesis occurs by tissue phase separation controlled by a bistable cell-state switch with suppressed oscillatory dynamics The tetrapod limb skeleton is initiated in unpatterned limb bud mesenchyme by the formation of precartilage condensations. Here, based on time-lapse videographic analysis of a forming condensation in a high-density culture of chicken limb bud mesenchyme, we observe a phase transition to a more fluidized state for cells within spatial compacted foci (protocondensations that will progress to condensations), as reflected in their spatial confinement, cell-substratum interaction and speed of motion. Previous work showed that galectin-8 and galectin-1A, two proteins of the galactoside-binding galectin family, are the earliest determinants of this process in the chicken limb bud, and that their interactions in forming skeletogenic patterns of condensations can be interpreted mathematically through a reaction-diffusion-adhesion framework. Based on this framework, we use an ordinary differential equation-based approach to analyze the core switching modality of the galectin reaction network and characterize the states of the network independent of the diffusive and adhesive arms of the patterning mechanism. We identify two steady states where the concentrations of both galectins are respectively, negligible, and very high. An explicit Lyapunov function shows that there are no periodic solutions. For sigmoidal galectin production terms, the model exhibits a bistable switch that arises from a monostable state via saddle-node bifurcation. Our model therefore predicts that the galectin network exists in low and high expression states separated in space or time without any intermediate states. This provides a causal basis for the observed outside vs. inside transition observed in the in vitro video data. We performed a quantitative analysis of the distribution of galectin-1A in cultures of condensing chick limb mesenchymal cells and found that the interior of the protocondensations had concentrations of this protein (compared to the immediate exterior) over and above that expected from its higher cell density, consistent with the models predictions. The galectin-based patterning network is thus suggested, on theoretical grounds, to incorporate a core switch independent of any spatial or temporal dynamics, that drives the chondrogenic cell state transition in limb skeletogenesis. Warmer temperatures result in maladaptive learning of sexual preferences O_LIEmpirical studies predicting the effects of the climate crisis on biodiversity loss that take into account learning ability and sexual selection are currently lacking.
C_LIO_LIUsing the African butterfly Bicyclus anynana, which shows strong phenotypic plasticity (i.e., polyphenism) in response to temperature, we find that learning produces a maladaptive mate preference under climate warming.
C_LIO_LIWe first modelled climate warming scenarios and then showed experimentally that as temperature becomes an unreliable cue to the onset of the dry season, adult butterflies displayed the wet season rather than the dry season form.
C_LIO_LIFemale learning further suppressed their innate, adaptive sexual preference for dry season males. Instead, females learned to prefer a phenotype transiently present during the seasonal transition.
C_LIO_LIFemale fertility and longevity were also indirectly affected by learning, likely reducing female fitness following climate warming.
C_LIO_LIOur results emphasize the importance of sexual selection, learning, and their fitness consequences for understanding (mal)adaptation of natural populations to climate warming.
C_LI Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. AlphaFold2-aware protein-DNA binding site prediction using graph transformer Protein-DNA interactions play crucial roles in the biological systems, and identifying protein-DNA binding sites is the first step for mechanistic understanding of various biological activities (such as transcription and repair) and designing novel drugs. How to accurately identify DNA-binding residues from only protein sequence remains a challenging task. Currently, most existing sequence-based methods only consider contextual features of the sequential neighbors, which are limited to capture spatial information. Based on the recent breakthrough in protein structure prediction by AlphaFold2, we propose an accurate predictor, GraphSite, for identifying DNA-binding residues based on the structural models predicted by AlphaFold2. Here, we convert the binding site prediction problem into a graph node classification task and employ a transformer-based variant model to take the protein structural information into account. By leveraging predicted protein structures and graph transformer, GraphSite substantially improves over the latest sequence-based and structure-based methods. The algorithm is further confirmed on the independent test set of 181 proteins, where GraphSite surpasses the state-of-the-art structure-based method by 16.4% in AUPR and 11.2% in MCC, respectively. We provide the datasets, the predicted structures, and the source codes along with the pre-trained models of GraphSite at https://github.com/biomed-AI/GraphSite. The GraphSite web server is freely available at https://biomed.nscc-gz.cn/apps/GraphSite. Ending animal agriculture would stabilize greenhouse gas levels for 30 years and offset 70 percent of CO2 emissions this century Animal agriculture contributes significantly to global warming through ongoing emissions of the potent greenhouse gases methane and nitrous oxide, and displacement of biomass carbon on the land used to support livestock. However, because estimates of the magnitude of the effect of ending animal agriculture often focus on only one factor, the full potential benefit of a more radical change remains underappreciated. Here we quantify the full "climate opportunity cost" of current global livestock production, by modeling the combined, long-term effects of emission reductions and biomass recovery that would be unlocked by a phaseout of animal agriculture. We show that, even in the absence of any other emission reductions, persistent drops in atmospheric methane and nitrous oxide levels, and slower carbon dioxide accumulation, following a phaseout of livestock production would, through the end of the century, have the same cumulative effect on the warming potential of the atmosphere as a 25 gigaton per year reduction in anthropogenic CO2 emissions, providing half of the net emission reductions necessary to limit warming to 2{degrees}C. The magnitude and rapidity of these potential effects should place the reduction or elimination of animal agriculture at the forefront of strategies for averting disastrous climate change.

Significance StatementThe use of animals to produce food has a negative impact on the climate, but the benefits of a global switch to a plant based diet are underappreciated. We show that the global warming impact, through the rest of this century, of eliminating greenhouse gas emissions from livestock and allowing native ecosystems to regrow on the land currently used to house and feed livestock, would be equivalent to a 68% reduction in carbon dioxide emissions. We hope putting clearer numbers on the "climate opportunity cost" of our continued use of animals as food technology will help policymakers and the public properly prioritize dietary change as a climate defense strategy.

Declaration of Conflict of InterestPatrick Brown is the founder and CEO of Impossible Foods, a company developing alternatives to animals in food-production. Michael Eisen is an advisor to Impossible Foods. Both are shareholders in the company and thus stand to benefit financially from reduction of animal agriculture. The compound of baicalein, wogonin, and oroxylin-A inhibits EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. High-throughput analysis of single human cells reveals the complex nature of DNA replication timing control DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We developed an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes "in silico flow cytometry", a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyzed up to 2,437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression. C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here the interaction of enzymes fumarase FumB and aspartase AspA with the C4-DC transporters DcuA and DcuB of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography (mSPINE). DcuB interacted strongly with FumB and AspA, and DcuA with AspA. The fumB-dcuB and the dcuA-aspA genes encoding the respective proteins are known for their colocalization on the genome and the production of co-transcripts. The data consistently suggest the formation of DcuB/FumB, DcuB/AspA and DcuA/AspA metabolons in fumarate respiration for the uptake of L-malate, or L-aspartate, conversion to fumarate and excretion of succinate after reduction. The DcuA/AspA metabolon catalyzes L-Asp uptake and fumarate excretion in concerted action also to provide ammonia for nitrogen assimilation. Ecological interactions in breast cancer: Cell facilitation promotes growth and survival under drug pressure The interplay of positive and negative interactions between drug sensitive and resistant cells determines the efficacy of treatment in heterogeneous cancer cell populations. We developed isogenic estrogen receptor positive (ER+) breast cancer cell lineages sensitive and resistant to ribociclib-induced CDK4/6 inhibition to investigate cell interactions under selective pressure. During treatment with ribociclib alone or in combination with endocrine therapies, sensitive cells survive and proliferate better when grown together with resistant cells than when grown in monoculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays detected estrone, a less active metabolite, in medium taken from each lineage but detected highly active estradiol only from resistant cells. Because sensitive cells are more dependent on estrogen signaling than resistant cells, they benefit from the increase in local estradiol from resistant cells, leading to facilitated growth when grown under treatment in coculture. We developed a suite of mathematical models to quantify the strength of competition and facilitation between cell lines and evaluate the effects of treatments that modify these interactions. These analyses support the conclusion that resistant cell lineages facilitate growth of sensitive lineages during therapy through the increased production and sharing of local estradiol and themselves suffer from increased competition and suppression as a result. These models show that blocking facilitation has the potential to control both resistant and sensitive cell populations and inhibit the emergence of a refractory population. This combination of experiments and models shows that interactions within heterogeneous cancer cell populations can combine positive and negative interactions to shape the dynamics of resistance. Variation in Ubiquitin System Genes Creates Substrate-Specific Effects on Proteasomal Protein Degradation Ubiquitin-proteasome system (UPS) protein degradation regulates protein abundance and eliminates mis-folded and damaged proteins from eukaryotic cells. Variation in UPS activity influences numerous cellular and organismal phenotypes. However, to what extent such variation results from individual genetic differences is almost entirely unknown. Here, we developed a statistically powerful mapping approach to characterize the genetic basis of variation in UPS activity. Using the yeast Saccharomyces cerevisiae, we systematically mapped genetic influences on the N-end rule, a UPS pathway that recognizes N-degrons, degradation-promoting signals in protein N-termini. We identified 149 genomic loci that influence UPS activity across the complete set of N-degrons. Resolving four loci to individual causal nucleotides identified regulatory and missense variants in ubiquitin system genes whose products process (NTA1), recognize (UBR1 and DOA10), and ubiquitinate (UBC6) cellular proteins. Each of these genes contained multiple causal variants and several individual variants had substrate-specific effects on UPS activity. A cis-acting promoter variant that modulates UPS activity by altering UBR1 expression also alters the abundance of 36 proteins without affecting levels of the corresponding mRNAs. Our results demonstrate that natural genetic variation shapes the full sequence of molecular events in protein ubiquitination and implicate genetic influences on the UPS as a prominent source of post-translational variation in gene expression. Plucking a string or playing a G? Choice type impacts human reinforcement learning In reinforcement learning (RL) experiments, participants learn to make rewarding choices in response to different stimuli; RL models use outcomes to estimate stimulus-response values which change incrementally. RL models consider any response type indiscriminately, ranging from more concretely defined motor choices (e.g. pressing a key with the index finger), to more general choices that can be executed in a number of ways (e.g. getting dinner at the restaurant). But does the learning process vary as a function of the choice type? In Experiment 1, we show that it does: participants were slower and less accurate in learning correct choices of a general format compared to learning more concrete, motor actions. Using computational modeling, we show that two mechanisms contribute to this. First, there was evidence of irrelevant credit assignment: the values of motor actions interfered with the values of other choice dimensions, resulting in more incorrect choices when the correct response is not defined by a single motor action; second, information integration for relevant general choices was slower. In Experiment 2, we replicated and further extended the findings from Experiment 1, by showing that slowed learning was attributable to weaker working memory use, rather than slowed RL learning. In both experiments we ruled out the explanation that the difference in performance between two condition types was driven by difficulty/different levels of complexity. We conclude that defining a more abstract choice space used by multiple learning systems for credit assignment recruits executive resources, limiting how much such processes then contribute to fast learning. A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro in cancer cells and in vivo in mice Nano-based delivery systems have enhanced our ability to administer and target drugs and macromolecules to their targets. Oligonucleotide drugs have great therapeutic potential but often have off-target effects and stability issues. Therefore, they are often encapsulated in vesicles with targeting ligands such as antibodies (Ab) to deliver their cargo. Herein, we describe a novel, scalable and straightforward approach to producing functionalized vesicles called the "Functionalized Lipid Insertion Method," which differs from the older "Detergent-Dialysis Method." The latter method required excess detergent and extensive dialysis over many hours to produce the functionalized vesicles. With our method, only the functionalized lipid is detergent-solubilized during the engineering of the vesicle. The approach reduces the dialysis time, keeps the vesicles intact while orienting the targeting moieties of the functionalized lipid toward the outside of the vesicle. Pilot in vitro and in vivo experiments was performed to show the feasibility of our method. Dynamic light scattering (DLS) experiments suggested that the original vesicular structure was relatively unperturbed, and the functionalized lipid was inserted externally. Our approach efficiently delivered oligonucleotides and affected the function of liver cancer HepG2 cells. Furthermore, functionalized vesicles achieved targeted delivery of oligonucleotides in mice without inducing a significant innate immune response. The industrial and therapeutic significance and implications of functionalized vesicles produced by our method are also discussed. Additional experiments and analyses are recommended to bring out the full potential of this molecular delivery technology. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Empirical Transmit Field Bias Correction of T1w/T2w Myelin Maps T1-weighted divided by T2-weighted (T1w/T2w) myelin maps were initially developed for neuroanatomical analyses such as identifying cortical areas, but they are increasingly used in statistical comparisons across individuals and groups with other variables of interest. Existing T1w/T2w myelin maps contain radiofrequency transmit field (B1+) biases, which may be correlated with these variables of interest, leading to potentially spurious results. Here we propose two empirical methods for correcting these transmit field biases using either explicit measures of the transmit field or alternatively a 'pseudo-transmit' approach that is highly correlated with the transmit field at 3T. We find that the resulting corrected T1w/T2w myelin maps are both better neuroanatomical measures (e.g., for use in cross-species comparisons), and more appropriate for statistical comparisons of relative T1w/T2w differences across individuals and groups (e.g., sex, age, or body-mass-index) within a consistently acquired study at 3T. We recommend that investigators who use the T1w/T2w approach for mapping cortical myelin use these B1+ transmit field corrected myelin maps going forward. Plant-environment microscopy tracks interactions of Bacillus subtilis with plant roots across the entire rhizosphere Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants large volume of influence. Here, we present the development of 3D live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor made dual-illumination light-sheet system acquired scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3 in size with as good as 5 m resolution at a rate of up to one scan every 30 minutes. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed towards the root cap, the point "first contact", before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions. A combined human in silico and CRISPR/Cas9-mediated in vivo zebrafish based approach for supporting gene target validation in early drug discovery The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy. 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. Proton-driven alternating access in a spinster transporter, an emerging family of broad-specificity efflux pumps Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. We employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange. Furthermore, our resistance assays reveal substrate polyspecificity and HnSpns multidrug resistance (MDR) activity that underscore the previously unknown role of Spns proteins in MDR, beyond their activity in sphingolipid transport and signaling. Effects of substrate on shrimp growth, water quality and bacterial community in the biofloc system nursing Penaeus vannamei postlarvae This study aimed to investigate the effects of substrate on water quality, shrimp growth and bacterial community in the biofloc system with a salinity of 5{per thousand}. Two treatments, biofloc system with (sB) or without (nB) addition of elastic solid packing filler (nylon) as substrate, were set up. Penaeus vannamei postlarvae (PL, [~] stage 15) were stocked at a density of 4000 PL m-3 in each treatment with triplicates for a 28-days culture experiment, taking glucose as carbon source (C:N 15:1). Results showed that the survival rate (96.3{+/-}3.6%), FCR (0.76{+/-}0.06) and productivity (1.54{+/-}0.12 kg m-3) in sB treatment were significantly better than those in nB treatment (81.0{+/-}7.1%, 0.98{+/-}0.08 and 1.14{+/-}0.09 kg m-3, P<0.05). All water parameters were in the recommended ranges. Substrate showed significant effect on TAN, TSS, turbidity, biofloc volume, pH and carbonate alkalinity (P < 0.05). Actinobacteria (4.0-22.7%), Bacteroidetes (10.4-33.5%), Firmicutes (0.2-11.2%), Planctomycetes (4.0-14.9%) and Proteobacteria (29.4-59.0%) were the most dominant phyla for both treatments. However, the bacterial community in sB treatment showed to be significantly different from that in nB treatment (Jaccard distance 0.94{+/-}0.01, P=0.001). Substrate showed significant effects on Shannon, Heip, Pielou and Simpson index, as well as relative abundances of Actinobacteria, Bacteroidetes and Planctomycetes (P < 0.05). The results suggested that addition of substrate affected the shrimp growth, water quality and bacterial community in the biofloc system nursing P. vannamei PL with a 5{per thousand} salinity. 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. Identification of a Golgi-localized altORF peptide reveals a minimal Golgi targeting motif Prior work has identified signal sequences and motifs that are necessary and sufficient to target proteins to specific subcellular regions and organelles such as the plasma membrane, nucleus, Endoplasmic Reticulum, and mitochondria. In contrast, minimal sequence motifs that are sufficient for Golgi localization remain largely elusive. In this work, we identified a 37 amino acid alternative open reading frame (altORF) within the mRNA of the centromere protein, CENP-R. This altORF peptide localizes specifically to the cytoplasmic surface of the Golgi apparatus. Through mutational analysis, we identify a minimal 10 amino acid sequence and a critical cysteine residue that are necessary and sufficient for Golgi localization. Pharmacological perturbations suggest that this peptide is modified through palmitoylation to promote its localization. Together, our work defines a minimal sequence that is sufficient for Golgi targeting and provide a valuable Golgi marker for live cell imaging. Targeting MLL methyltransferases enhance the anti-tumor effects of PI3K inhibition in hormone receptor-positive breast cancer The high frequency of aberrant PI3K pathway activation in hormone receptor-positive (HR+) breast cancer has led to the development, clinical testing, and approval of the p110-selective PI3K inhibitor alpelisib. The limited clinical efficacy of alpelisib and other PI3K inhibitors is partially attributed to the functional antagonism between PI3K and estrogen receptor (ER) signaling, which is mitigated via combined PI3K inhibition and endocrine therapy. We and others have previously demonstrated a chromatin-associated mechanism by which PI3K supports cancer development and antagonizes ER signaling through the modulation of H3K4 methylation. Here we show that inhibition of the H3K4 histone methyltransferase MLL1 in combination with PI3K inhibition impairs HR+ breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces AKT effector signaling and H3K4 methylation, MLL1 inhibition increases PI3K effector signaling and upregulates the expression of receptor tyrosine kinase signaling cascades upstream of and including AKT. These data reveal a feedback loop between MLL1 and AKT in which MLL1 inhibition reactivates AKT. We additionally show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro and in vivo models of HR+ breast cancer, which is enhanced by the additional genetic ablation of the H3K4 methyltransferase and AKT target MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and may support the preclinical development and testing of pan-MLL inhibitors.

Significance StatementPharmacological inhibition of PI3K provides limited efficacy in PIK3CA-mutated, HR+ breast cancers. Here the authors leverage PI3K/AKT-driven chromatin modification to identify MLL histone methyltransferases as a therapeutic target. Dual PI3K and MLL inhibition synergize to reduce clonogenicity and cell proliferation while enhancing apoptosis in in vitro models, and induces tumor regression in xenograft models of PI3K-activated, HR+ breast cancer. Furthermore, MLL1 inhibition reveals a feedback loop leading to AKT hyperactivation, which is relieved with combined PI3K/MLL inhibition. These findings demonstrate the utility of MLL inhibitors for the treatment of some solid cancers, as patients with HR+ breast cancer characterized by PIK3CA mutation may derive clinical benefit from combined PI3K/MLL inhibition. Muscle Activation Profile During Perturbed Walking is Modulated According to Body State During an unexpected loss of balance, avoiding a fall requires people to readjust their footing rapidly and effectively. We investigated the activation patterns of the ankle and knee muscles, and muscle fiber type recruitment resulting from unannounced, medio-lateral (i.e., right/left) horizontal-surface walking perturbations in twenty healthy adults (27.00{+/-}2.79 years, 10 females). Surface electromyography (sEMG) total spectral power for specific frequency bands (40-60Hz, 60-150Hz, 150-250Hz, 250-400Hz and 400-1000Hz), from tibialis anterior (TA) and vastus lateralis (VL) muscles were analyzed. Compared to non-perturbed walking, we found a significant increase in the total spectral power of lower-extremity muscles during the first 3 seconds after perturbation. When two feet were on the ground in time of perturbation we found a different muscle fiber type recruitment pattern between VL and TA muscles. This was not significant for perturbations implemented when one foot was on the ground. Our findings suggest that muscle operating frequency is modulated in real time to fit body state and somatosensory input in the context of functional goal requirements such as a rapid change of footing in response to unexpected loss of balance in single and double-support phases of gait.

New & NoteworthyTo study muscle spectral profiles in response to loss of balance, we investigated the dynamics of muscle spectral power changes, across different frequency bands after unannounced mechanical perturbations during walking. We showed increased activation of high-frequency motor units of the lower-limb muscles, subside 3 seconds after perturbation. Differences in power increase of specific frequency bands suggest that muscle activation is modulated in real time to fit body state in the context of functional goal requirements.