Quercetin handles cellular oxidant / antioxidant systems and mitigates immunosenescence hallmarks in human PBMCs: an in vitro study Immunosenescence, oxidative stress, and low vaccine efficacy are important symptoms of aging. The goal of our study was to see if quercetin had anti-aging and stimulating effects on PBMC immune cells in vitro. In the presence of concanavalin a, PBMCs were isolated from healthy elderly and young people and cultured in a complete RPMI-1640 medium supplemented with quercetin. Cell proliferation was assessed using the MTT colorimetric assay after a 48-hour incubation period. Spectrophophotometric assays were used to assess oxidative biomarkers (PC, MDA, and GSH). The ELISA method was used to determine the amount of IL-2 released. A Griess reagent was used to investigate iNOS activity. When compared to young control cells, aged PBMCs had lower proliferation potency, lower IL-2 and NO release, and higher MDA and PC levels. Importantly, quercetin-treated aged PBMCs have a high proliferative response comparable to young cells, restored iNOS activity, and increased levels of GSH antioxidant defences. In comparison to untreated aged PBMCs, treated PBMCs have lower lipo-oxidative damage but higher PC levels. Quercetin may be used as a promising dietary vaccinal adjuvant in the elderly, it has significant effects in reducing immunosenescence hallmarks, as well as mitigating the lipo-oxidative stress in PBMCs cells.

Practical abstractQuercetin is a common dietary polyphenol found in fruits and vegetables that is well known for its antioxidant activity in radical scavenging and anticancer properties characterized by immune system stimulation. In vitro, quercetin boosts immune responses in the elderly and reduces immunosenescence symptoms, according to our findings. As a result, this molecule could be a safe and promising vaccine adjuvant for boosting immunity and reducing aging complications in immune cells as well as reducing oxidative stress in PBMC cells.
Beneficial effects of novel strains of Aureobasidium pullulans produced 1,3-1,6 β-glucans on non-esterified fatty acid levels in diabetic KKAy mice Obesity, metabolic syndrome, associated lipotoxicity and its cascade of events contribute to the majority of the burden related to non-communicable diseases globally. Preventive lifestyle changes aside, several beneficial effects have been reported in type II diabetes mellitus and dyslipidaemia patients with biological response modifier glucans (BRMG) produced as an exopolysaccharide by Aureobasidium pullulans. In this study, we compared two strains (AFO-202 and N-163) that produce beta glucans in alleviating lipotoxicity. This study was performed in obese diabetic mice model of KK-Ay mice, in four groups with six subjects in each group - Group 1: sacrificed on Day 0 for baseline values; Group 2: control (drinking water); Group 3: AFO-202 beta glucan--200 mg/kg/day; Group 4: N-163 beta glucan--300 mg/kg/day. The animals in groups 2-4 had the test solutions administered by gavage once daily for 28 consecutive days. Biochemical analyses were conducted of blood glucose, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol and non-esterified fatty acids (NEFA). Group 4 (N-163) had the lowest NEFA levels, as compared to the other groups, and marginally decreased triglyceride levels. The groups had no significant differences in blood glucose, HbA1c, triglycerides, or LDL and HDL cholesterol. N-163 produced by A. pullulans decreased NEFA in a diabetic mice model in 28 days. These results, although modest, warrant further in-depth research into lipotoxicity and associated inflammatory cascades in both healthy and disease affected subjects to develop novel strategies for prevention and management.
Social behavioral profiling by unsupervised deep learning reveals a stimulative effect of dopamine D3 agonists on zebrafish sociality It has been a major challenge to systematically evaluate and compare how pharmacological perturbations influence social behavioral outcomes. Although some pharmacological agents are known to alter social behavior, precise description and quantification of such effects have proven difficult. The complexity of brain functions regulating sociality makes it challenging to predict drug effects on social behavior without testing in live animals, and most existing behavioral assays are low-throughput and provide only unidimensional readouts of social function. To achieve richer characterization of drug effects on sociality, we developed a scalable social behavioral assay for zebrafish named ZeChat based on unsupervised deep learning. High-dimensional and dynamic social behavioral phenotypes are automatically classified using this method. By screening a neuroactive compound library, we found that different classes of chemicals evoke distinct patterns of social behavioral fingerprints. By examining these patterns, we discovered that dopamine D3 agonists possess a social stimulative effect on zebrafish. The D3 agonists pramipexole, piribedil, and 7-hydroxy-DPAT-HBr rescued social deficits in a valproic acid-induced zebrafish autism model. The ZeChat platform provides a promising approach for dissecting the pharmacology of social behavior and discovering novel social-modulatory compounds. 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. Subthreshold repetitive transcranial magnetic stimulation drives structural synaptic plasticity in the young and aged motor cortex. BackgroundRepetitive transcranial magnetic stimulation (rTMS) is a non-invasive tool commonly used to drive neural plasticity in the young adult and aged brain. Recent data from mouse models have shown that even at subthreshold intensities (0.12 Tesla), rTMS can drive neuronal and glial plasticity in the motor cortex. However, the physiological mechanisms underlying subthreshold rTMS induced plasticity and whether these are altered with normal ageing are unclear.

ObjectiveTo assess the effect of subthreshold rTMS, using the intermittent theta burst stimulation (iTBS) protocol on structural synaptic plasticity in the mouse motor cortex of young and aged mice.

MethodsLongitudinal in vivo 2-photon microscopy was used to measure changes to the structural plasticity of pyramidal neuron dendritic spines in the motor cortex following a single train of subthreshold rTMS (in young adult and aged animals) or the same rTMS train administered on 4 consecutive days (in young adult animals only). Data were analysed with Bayesian hierarchical generalized linear regression models and interpreted with the aid of Bayes Factors (BF).

ResultsWe found strong evidence (BF>10) that subthreshold rTMS altered the rate of dendritic spine losses and gains, dependent on the number of stimulation sessions and that a single session of subthreshold rTMS was effective in driving structural synaptic plasticity in both young adult and aged mice.

ConclusionThese findings provide further evidence that rTMS drives synaptic plasticity in the brain and uncovers structural synaptic plasticity as a key mechanism of subthreshold rTMS induced plasticity. Ecology of active viruses and their bacterial hosts in frozen Arctic peat soil revealed with H218O stable isotope probing metagenomics Winter carbon loss in northern ecosystems is estimated to be greater than the average growing season carbon uptake. However, most ecosystem carbon measurements neglect winter months since carbon losses (primarily driven by microbial decomposers) are assumed to be negligible at low temperatures. We used stable isotope probing (SIP) targeted metagenomics to reveal the genomic potential of active soil microbial populations under winter conditions, with an emphasis on viruses and virus-host dynamics. Peat soils from the Bonanza Creek LTER site in Alaska were incubated under subzero anoxic conditions with H218O for 184 and 370 days. We identified 46 bacterial populations (MAGs; spanning 9 bacterial phyla) and 243 viral populations (vOTUs) that actively took up 18O and produced significant CO2 throughout the incubation. Active hosts, predicted for 33% of the active vOTUs, were some of the most abundant MAGs and capable of fermentation and organic matter degradation. Approximately three-quarters of the active vOTUs carried auxiliary metabolic genes that spanned five functional categories, including carbon utilization, highlighting the potential impact of viruses in this peat soils microbial biogeochemistry. These results illustrate significant bacterial and viral activity and interactions occur in frozen soils, revealing viruses are a major community-structuring agent throughout winter months. Evaluating the Anti-aging Effects of Chinese Herbal Medicine Rhodiola rosea L. in Cultured Keratinocytes Among the many health benefits Chinese herbal medicine (CHM) presents, anti-aging is of special interest (Shen, Jiang, Yang, Wang, & Zhu, 2017; Zhao & Luo, 2017). Reported to possess anti-aging effects, the CHM Rhodiola rosea L., known colloquially as the "golden root", has been widely incorporated in various drinks, daily supplements, and even cosmetics. This study investigates the effects of commercial R. rosea extracts and natural R. rosea roots on preventing UV-induced photoaging of the skin and correlates such effects with the composition of active ingredients in the extracts. To simulate the photoaging process, drug treated HaCaT cells were exposed to UVA and UVB radiation. The pharmacological anti-aging effects of R. rosea extracts were evaluated qualitatively and quantitatively through confocal immunofluorescence images with {gamma}-H2A.X marker and telomerase activity assay (Telo TAGGG Telomerase PCR-ELISA). Preparatory thin layer chromatography and high-performance liquid chromatography were performed to isolate and quantify active ingredients. Cultured HaCaT cells showed morphological change after exposure to both UVA (>15.0 J) and UVB (>2000 mJ). The photoaging of keratinocytes was rescued by pretreating cells with R. rosea extracts as well as salidroside and rosavin active ingredients (P < 0.05). R. rosea-treated cells were characterized by increased telomerase activity and fewer {gamma}-H2A.X foci compared to that of the control. Extracts with better preventative effects contained higher salidroside and rosavin content. The findings in this study reaffirm R. roseas efficacy as an anti-aging remedy and provide a basis for CHMs integration into the mainstream of global healthcare. Transmembrane helices are an overlooked and evolutionarily conserved source of major histocompatibility complex class I and II epitopes Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most haplotypes of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHCI and MHC-II. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells respond more to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs therefore are less prone to escape mutations that enable pathogens to evade T cell responses. The SLIM1 transcription factor is required for arsenic resistance in Arabidopsis thaliana The transcriptional regulators of arsenic-induced gene expression remain largely unknown. Sulfur assimilation is tightly linked with arsenic detoxification. Here we report that mutant alleles in the SLIM1 transcription factor are substantially more sensitive to arsenic than cadmium. Arsenic treatment caused high levels of oxidative stress in the slim1 mutants, and slim1 alleles were impaired in both thiol and sulfate accumulation. We further found enhanced arsenic accumulation in roots of slim1 mutants. Transcriptome analyses indicate an important role for SLIM1 in arsenic-induced tolerance mechanisms. The present study identifies the SLIM1 transcription factor as an essential component in arsenic tolerance and arsenic-induced gene expression. Our results suggest that the severe arsenic sensitivity of the slim1 mutants is caused by altered redox status. Mouse lemur, a new primate model to investigate cardiac pacemaker activity. BackgroundGray mouse lemurs (Microcebus murinus) are one of the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since Microcebus murinus replicates similar senescence processes of humans, it constitutes a useful model also for studying cardiovascular dysfunctions. However, its cardiac physiology is unknown. Thus, we investigated sinoatrial node (SAN) pacemaker activity of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.

Methods and ResultsWe recorded the heart automaticity in freely moving mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of electrical impulses in the SAN tissue. Conversely, their ventricular depolarization is more similar to large mammals. Mouse lemur SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (ICa,L) and T-type (ICa,T) calcium currents. Moreover, mouse lemur SAN myocytes display spontaneous ryanodine receptor (RyR)-dependent Ca2+ transients. Using muscle-derived stem cells (MDSCs) from the Microcebus murinus skeletal muscle, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).

ConclusionsIn conclusion, our characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans, and for pharmacologic testing of molecules that could assist in modulating age-related dysfunctions of automaticity. Anaerobic Thermophilic Co-digestion of Sugarcane Biorefinery Residues Enhances Biogas Production This is a pioneer study evaluating the methane (CH4) production potential from residues of integrated 1st (vinasse and filter cake) and 2nd (deacetylation pretreatment liquor from straw) generation (1G2G) sugarcane biorefinery, providing a fully chemical characterization of them and their relation with the anaerobic digestion (AD) process. Small-scale assays provided fundamentals for basing the co-digestion optimization by assessing the optimal co-substrates synergistic conditions. Biochemical Methane Potential (BMP) tests showed co-digestion enhanced CH4 yield of isolated substrates, reaching up to 605 NmLCH4 gVS-1. The association of vinasse and deacetylation liquor as co-substrates increased the BMP by ~38% mostly by nutritionally benefiting the methanogenic activity. The kinetic analysis confirmed that the deacetylation liquor was the co-substrate responsible for improving the CH4 production in the co-digestion systems due to the highest CH4 conversion rate. The alkaline characteristic of the liquor (pH~12) also prevented alkalizing from being added to the co-digestion, an input that normally makes the process economically unfeasible to implement on an industrial scale due to the large quantities required for buffering the reactor. The filter cake had the lowest BMP (262 NmLCH4 gVS-1) and digestibility ([&le;] 40%), further limited by the required stirring to improve the mass transfer of biochemical reactions. The present study drives towards more sustainable use of vinasse, the most voluminous waste from the sugarcane industry, and lignin-rich residues derived from pre-treatment alkaline methods, aiming at an energy-efficient utilization, by at least 16% when compared to the traditional vinasse AD. The experimental and modeling elements from this work indicated the lignin-rich liquor is the main responsible for putting the co-digestion as a disruptive technological arrangement within the 1G2G sugarcane biorefineries, reinforcing the biogas production as the hub of the bioeconomy in the agroindustrial sector. Single amino-acid mutation in a Drosophila melanogaster ribosomal protein: an insight in uL11 transcriptional activity The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co- localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. RNA- seq analysis from wing imaginal discs shows enrichment in the GO categories "glutathione metabolism" for up-regulated genes in both uL11K3A and uL11K3Y mutants and "regulation of transcription" for down-regulated genes in uL11K3A only. Analysis of the cis-regulatory sequences of these genes suggests that uL11 might regulate transcription of target genes in concert with the couple of transcription factors Mad/Med that mediate response to the Bone Morphogenetic Protein (BMP) signaling pathway. Entorhinal cortex epigenome-wide association study highlights four novel loci showing differential methylation in Alzheimer's disease BackgroundStudies on DNA methylation (DNAm) in Alzheimers disease (AD) have recently highlighted several genomic loci showing association with disease onset and progression.

MethodsHere, we conducted an epigenome-wide association study (EWAS) using DNAm profiles in entorhinal cortex (EC) from 149 AD patients and control brains and combined these with two previously published EC datasets by meta-analysis (total n=337).

ResultsWe identified 12 cytosine-phosphate-guanine (CpG) sites showing epigenome-wide significant association with either case-control status or Braaks tau-staging. Four of these CpGs, located in proximity to CNFN/LIPE, TENT5A, PALD1/PRF1, and DIRAS1, represent novel findings. Integrating DNAm levels with RNA sequencing-based mRNA expression data generated in the same individuals showed significant DNAm-mRNA correlations for 6 of the 12 significant CpGs. Lastly, by calculating rates of epigenetic age acceleration using two recently proposed "epigenetic clock" estimators we found a significant association with accelerated epigenetic aging in AD patients vs. controls.

ConclusionIn summary, our study represents the hitherto most comprehensive EWAS in AD using EC and highlights several novel differentially methylated loci with potential effects on gene expression. Vectofusin-1 based T-cell transduction approach for generating murine chimeric antigen receptor T-cells for cancer. Gene transfer into human and murine T-cells using viral-based approaches has several promising therapeutic applications including the production of chimeric antigen receptor T-cell (CAR-T) therapy. The generation of murine CAR-T is paramount to test and validate immunocompetent mouse models for CAR-T therapy. Several viral transduction enhancers already exist for gene therapy with few limitations. In this study, we tested vectofusin-1, a short cationic peptide, as a soluble transduction enhancer for gammaretroviral transduction for the generation of anti-CD19 murine CAR-T. We found that in comparison to Retronectin, Vectofusin-1 is an equally optimal transduction enhancer for the generation of murine CAR-T cells. The snoGloBe interaction predictor reveals a broad spectrum of C/D snoRNA RNA targets Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2-O-ribose methylation. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA-RNA interaction datasets detect many snoRNAs binding messenger RNA, but are limited by specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. SnoGloBe is also an excellent tool for discovering viral RNA targets, as shown by its capacity to identify snoRNAs targeting the heavily methylated SARS-CoV-2 RNA. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function. Glucose inhibits haemostasis and accelerates diet-induced hyperlipidaemia in zebrafish larvae Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals. A platform of patient-derived microtumors identifies therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment. The isoforms of pyruvate kinase act as nutrient sensors for the β-cell KATP channel Insulin release from pancreatic {beta}-cells is triggered by the metabolism-dependent closure of ATP-sensitive K+ (KATP) channels. Here, we used isoform-specific deletion of pyruvate kinase (PK) to identify the unique roles of PKm1 and PKm2 in the {beta}-cell nutrient response. In the absence of PK activity, cytosolic ATP/ADP was insufficient to close KATP channels. Both PK isoforms were found on the plasma membrane, where they endow KATP channels with nutrient sensitivity and specificity. Although PKm1 accounts for >90% of cellular activity, PKm2 controls glucosedependent KATP closure. Amino acids, which generate mitochondrial phosphoenolpyruvate (PEP) without allosterically activating PKm2, depend on PKm1 to raise ATP/ADP and close KATP channels. Shifting the {beta}-cell from PKm2 to PKm1 correspondingly increased amino acid sensitivity and boosted secretory output. In addition to these membrane depolarizing "on-switches," {beta}-cell deletion of PCK2 exposed a mitochondrial PEP-dependent "off-switch" that facilitates Ca2+ extrusion. Restricting PCK2 prolonged Ca2+ influx and increased insulin secretion. In summary, it is not the KATP channel alone, but the PK-KATP microcompartment that confers nutrient sensitivity to the {beta}-cell plasma membrane. The differential response of the PK isoforms to the glycolytic and mitochondrial sources of PEP influences {beta}-cell nutrient preference, and controls the oscillatory cycle regulating insulin secretion. Intervene or wait? A model evaluating the timing of intervention in conservation conflicts adaptive management under uncertainty. The timing of biodiversity managers interventions can be critical to the success of conservation, especially in situations of conflict between conservation objectives and human livelihood, i.e., conservation conflicts. Given the uncertainty associated with complex social-ecological systems and the potentially irreversible consequences of delayed action for biodiversity and livelihoods, managers tend to simply intervene as soon as possible by precaution. However, refraining from intervening when the situation allows it can be beneficial, notably by saving critical management resources. Here, we introduce a strategy for managers to decide, based on monitoring, whether intervention is required or if waiting is possible. This study evaluates the performance of this waiting strategy compared to a strategy of unconditional intervention at every opportunity. We built an individual-based model of conservation conflict between a manager aiming to conserve an animal population and farmers aiming to maximize yield by protecting their crop from wildlife damage. We then simulated a budget-constrained adaptive management over time applying each strategy, while accounting for uncertainty around population dynamics and around decision-making of managers and farmers. Our results showed that when the decision for the manager to intervene was based on a prediction of population size trajectory, the waiting strategy performed at least as well as unconditional intervention while also allowing managers to save resources by avoiding unnecessary interventions. Under difficult budgetary constraints on managers, this waiting strategy ensured as high yields as unconditional intervention while significantly improving conservation outcomes by compensating managers lack of resources with the benefits accrued over waiting periods. This suggests that waiting strategies are worth considering in conservation conflicts, as they can facilitate equitable management with a more efficient use of management resources, which are often limiting in biodiversity conservation. 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. Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA BackgroundNon-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear.

MethodsUsing network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPAR agonist, fenofibrate, by MIR20B and the synergic effect of combination of fenofibrate with anti-MIR20B.

ResultsWe revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)- treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In MIR20B-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of MIR20B significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-MIR20B exhibited the synergic effect on improvement of NAFLD in MCD-fed mice.

ConclusionsTaken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD.

FundingThis research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940), and the Future-leading Project Research Fund (1.210034.01) of UNIST. 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. Ubiquity and origins of structural maintenance of chromosomes (SMC) proteins in eukaryotes Structural maintenance of chromosomes (SMC) protein complexes are common in Bacteria, Archaea, and Eukaryota. SMC proteins, together with the proteins related to SMC (SMC-related proteins), constitute a superfamily of ATPases. Bacteria/Archaea and Eukaryotes are distinctive from one another in terms of the repertory of SMC proteins. A single type of SMC protein is dimerized in the bacterial and archaeal complexes, whereas eukaryotes possess six distinct SMC subfamilies (SMC1-6), constituting three heterodimeric complexes, namely cohesin, condensin, and SMC5/6 complex. Thus, to bridge the homodimeric SMC complexes in Bacteria and Archaea to the heterodimeric SMC complexes in Eukaryota, we need to invoke multiple duplications of an SMC gene followed by functional divergence. However, to our knowledge, the evolution of the SMC proteins in Eukaryota had not been examined for more than a decade. In this study, we reexamined the ubiquity of SMC1-6 in phylogenetically diverse eukaryotes that cover the major eukaryotic taxonomic groups recognized to date and provide two novel insights into the SMC evolution in eukaryotes. First, multiple secondary losses of SMC5 and SMC6 occurred in the eukaryotic evolution. Second, the SMC proteins constituting cohesin and condensin (i.e., SMC1-4), and SMC5 and SMC6 were derived from closely related but distinct ancestral proteins. Based on the above-mentioned findings, we discuss how SMC1-6 have diverged from the archaeal homologs. Theory and Simulations of condensin mediated loop extrusion in DNA Condensation of hundreds of mega-base-pair-long human chromosomes in a small nuclear volume is a spectacular biological phenomenon. This process is driven by the formation of chromosome loops. The ATP consuming motor, condensin, interacts with chromatin segments to actively extrude loops. Motivated by real-time imaging of loop extrusion (LE), we created an analytically solvable model, predicting the LE velocity and step size distribution as a function of external load. The theory fits the available experimental data quantitatively, and suggests that condensin must undergo a large conformational change, induced by ATP binding, bringing distant parts of the motor to proximity. Simulations using a simple model confirm that the motor transitions between an open and a closed state in order to extrude loops by a scrunching mechanism, similar to that proposed in DNA bubble formation during bacterial transcription. Changes in the orientation of the motor domains are transmitted over ~ 50 nm, connecting the motor head and the hinge, thus providing an allosteric basis for LE. Activation of the type 3 secretion system of enteropathogenic E. coli leads to remodeling of its membrane composition and function The cell envelope of gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterial-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic E. coli (EPEC). The EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in bacterial envelope upon attachment to host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism towards the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase pldA and the acyltransferase ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant to abnormal synthesis of O-antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacteria cell size and Zeta potential, and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild type EPEC to the antibiotic.

ImportanceThe characterization of EPEC membrane lipid metabolism upon attachment to host is an important step towards a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent life style. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and show that it is associated with increased cell permeability and higher sensitivity to vancomycin.

To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system. A network approach to genetic circuit designs As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information.