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In this study, a novel KOH-activated biochar modified with Mg

The YTH N6-methyladenosine RNA binding proteins (YTHDFs) mediate the functional effects of N6-methyladenosine (m6A) on RNA. Recently, a report proposed that all YTHDFs work redundantly to facilitate RNA decay, raising questions about the exact functions of individual YTHDFs, especially YTHDF1 and YTHDF2. We show that YTHDF1 and YTHDF2 differ in their low-complexity domains (LCDs) and exhibit different behaviors in condensate formation and subsequent physiological functions. Biologically, we also find that the global stabilization of RNA after depletion of all YTHDFs is driven by increased P-body formation and is not strictly m6A dependent.

Accurate and large-scale screening of infected individuals has proven to be an effective means to control the spread of COVID-19. Currently, many assays have been developed to meet the huge testing requirements and the availability of diverse detection settings. However, few methods emphasize the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Herein, we developed a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (ORF1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 µL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab clinical samples showed 100% negative predictive agreement (NPA) and 97.14% positive predictive agreement (PPA). Expectantly, this developed platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.

Apical sodium-dependent bile acid transporter (ASBT) mediates the uptake of bile acids from the ileum lumen into enterocytes and presents a potential target for the treatment of several metabolic diseases, including type 2 diabetes. It has been proposed that the underlying mechanism for transport by ASBT is an elevator-style alternating-access model, which was deduced mainly by comparing high-resolution structures of two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii) in different conformations. However, one important issue is that the only outward-facing structure (PDB entry 4n7x) was obtained with an Na+-binding site mutant of ASBTYf, which severely cripples its transport function, and therefore the physiological relevance of the conformation in PDB entry 4n7x requires further careful evaluation. Here, another crystal structure is reported of ASBTYf that was captured in a state closely resembling the conformation in PDB entry 4n7x using an engineered disulfide bridge. The introduced cysteine mutations avoided any proposed Na+- or substrate-binding residues, and the resulting mutant retained both structural and functional integrity and behaved similarly to wild-type ASBTYf. These data support the hypothesis that the PDB entry 4n7x-like structure represents a functional outward-facing conformation of ASBTYf in its transport cycle.

Apical sodium-dependent bile acid transporter (ASBT) retrieves bile acids from the small intestine and plays a pivotal role in enterohepatic circulation. Currently, high-resolution structures are available for two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii), from which an elevator-style alternating-access mechanism has been proposed for substrate transport. A key concept in this model is that the substrate binds to the central cavity of the transporter so that the elevator-like motion can expose the bound substrate alternatingly to either side of the membrane during a transport cycle. However, no structure of an ASBT has been solved with a substrate bound in its central cavity, so how a substrate binds to ASBT remains to be defined. In this study, molecular docking, structure determination and functional analysis were combined to define and validate the details of substrate binding in ASBTYf. The findings provide coherent evidence to provide a clearer picture of how the substrate binds in the central cavity of ASBTYf that fits the alternating-access model.

Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.

Dexamethasone augments mitochondrial protein abundance. The translocase of the outer membrane (Tom) of mitochondria plays a major role in importing largely cytosolically synthesized proteins into mitochondria. We hypothesize that dexamethasone upregulates the Tom transport system, leading to increase of mitochondrial protein localization. Tom20 and Tom70 are the two major subunits. Dexamethasone increased Tom20 and Tom70 mRNA levels by 53 ± 11% and 25 ± 9% and mitochondrial protein abundance by 27 ± 7% and 25 ± 4% (p

Significance The TDP43 RNA binding protein is frequently aggregated in the brain tissue of patients suffering from neurodegenerative diseases. Human genetic studies of patients suffering from ALS have identified scores of missense mutations clustered within a localized region of the TDP43 protein. This region is of low sequence complexity and has been thought to exist in a state of structural disorder under conditions of proper TDP43 function. The present study gives evidence that the low complexity domain of TDP43 self-associates into a specific structural conformation that may be important to its normal biological function. Unlike prototypic low complexity domains, that of TDP43 is methionine-rich. Evidence is presented suggestive of the utility of these methionine residues in oxidation-mediated regulation of TDP43 function., A methionine-rich low complexity (LC) domain is found within a C-terminal region of the TDP43 RNA-binding protein. Self-association of this domain leads to the formation of labile cross-β polymers and liquid-like droplets. Treatment with H2O2 caused phenomena of methionine oxidation and droplet melting that were reversed upon exposure of the oxidized protein to methionine sulfoxide reductase enzymes. Morphological features of the cross-β polymers were revealed by H2O2-mediated footprinting. Equivalent TDP43 LC domain footprints were observed in polymerized hydrogels, liquid-like droplets, and living cells. The ability of H2O2 to impede cross-β polymerization was abrogated by the prominent M337V amyotrophic lateral sclerosis-causing mutation. These observations may offer insight into the biological role of TDP43 in facilitating synapse-localized translation as well as aberrant aggregation of the protein in neurodegenerative diseases.

The incidence rate of thyroid carcinoma, especially papillary thyroid carcinoma (PTC), has increased significantly over time. As a primary pathway for metastasis, the lymphatic system is an important prognostic factor for PTC patients. Although the metabolic changes in PTC patients have been investigated in extensive studies, few studies focused on the whole blood metabolic profiling of PTC patients. In this study, we investigated the 1H NMR-based metabolic profiles of whole blood samples that were obtained from healthy individuals and PTC patients, with or without lymph node metastasis. The estimation of the predictive potential of metabolites was evaluated using multivariate statistical analyses, which revealed that the whole blood carries information that is sufficient for distinguishing between PTC patients and healthy individuals. However, PTC patients were not well classified as positive or negative according to the lymph nodes. We did not find a metabolite that could discriminate the presence of lymph node metastasis. Further studies with larger sample sizes are needed to elucidate significant metabolites to indicate the presence of lymph node metastasis in patients with PTC.

The objective of this study was to investigate whether computed tomography texture analysis can be used to differentiate papillary renal cell carcinoma (PRCC) subtypes. Sixty-two PRCC tumors were retrospectively evaluated, with 30 type 1 tumors and 32 type 2 tumors. Texture parameters quantified from three-phase contrast-enhanced CT images were compared with least absolute shrinkage and selection operator (LASSO) regression. Receiver operating characteristic (ROC) analysis was performed, and the area under the ROC curve (AUC) was calculated for each parameter. The selected texture parameters of each phase were used to generate support vector machine (SVM) classifiers. Decision curve analysis (DCA) of the classification was performed. The two texture parameters with the top two AUC values were – 333–7 Correlation (AUC = 0.772) and 45–7 Entropy (AUC = 0.753) in the corticomedullary phase, 333–4 Correlation (AUC = 0.832) and 45–7 Entropy (AUC = 0.841) in the nephrographic phase, and 135–7 Entropy (AUC = 0.858) and – 333–1 InformationMeasureCorr2 (AUC = 0.849) in the excretory phase. Entropy and Correlation have a high correlation with the two types of PRCC and are increased in type 2 PRCC. A model incorporating the texture parameters with the top two AUC values in each phase produced an AUC of 0.922 with an accuracy of 84% (sensitivity = 89% and specificity = 80%). The nephrographic-phase model and the model combining the texture parameters of the three phases can differentiate the two types with the largest net benefit. Computed tomography texture analysis can be used to distinguish type 2 PRCC from type 1 with high accuracy, which may be clinically important.

Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported. The core of CUT-LAMP is the engineering of the forward or backward inner primer in the target-independent region, which makes the LAMP products contain a protospacer adjacent motif (PAM) site for the CRISPR/Cas9 recognition. For the CUT-LAMP reaction, cross-contamination can be efficiently cleaved by the corresponding Cas9/sgRNA, but the target gene can get rid of digestion due to the lack of a PAM site near the recognition region. CUT-LAMP shows impressive contamination resistance but does not significantly increase procedure complexity; thus, it represents a simple and versatile toolkit facilitating the adoption by open- and closed-tube detection format.

The recently reported freezing-based labeling method for constructing DNA-AuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling. However, successful labeling was restored by simply increasing the poly(A)-base number. Building on these discoveries, we developed three kinds of AuNP-based bioprobes, DNA-AuNP, RNA-AuNP, and DNA-enzyme-AuNP, using the freezing-based labeling method. This method was completed in a single mixing step with no need for thiol modification, representing one of the most convenient and lowest cost AuNP bioprobe labeling techniques ever reported. In addition, the resulting AuNP bioprobes were further used to advance CRISPR-based diagnostics through the development of user-friendly colorimetric, fluorescence, and lateral flow detection strategies.

Gold-nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective posthybridization manipulations for genomic analysis. Here, we present a new design for a colorimetric gene-sensing platform based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 h. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S rDNA or 16S rRNA. A CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity, thus, demonstrating its use as a robust next-generation gene detection platform.

Background: G0S2 was initially identified in blood mononuclear cells after the induction of cell cycle progression. The translation of G0S2 produces a small basic protein 103 amino acids in length. Initially, G0S2 was thought to mediate re-entry into the cell cycle by promoting the transition from the G0 to the G1 phase. Recent studies have identified roles for G0S2 in cancer, inflammation, and various metabolic processes. In lipid metabolism, G0S2 binds to ATGL and inhibits lipolysis. G0S2 also binds specifically to BCL-2 and inhibits the formation of the antiapoptotic body BCL-2/Bax, which in turn favors apoptosis. Whether there are other mechanisms and the proteins involved in these processes are not known; therefore, we used proteomics to identify differentially expressed proteins related to G0S2.Methods: G0S2-overexpressing adenovirus was injected into C57/BJL mice, the livers were collected, and proteins were identified by mass spectrometry. The identified proteins were analyzed by Gene Ontology (GO) and KEGG.Results: In total, 3765 liver proteins were identified, of which 3745 were quantified. The expression of 320 proteins was found to be upregulated, and that of 101 proteins was downregulated. These results indicated that G0S2 overexpression significantly altered the proteomic profile. There were 63 differential proteins involved in apoptosis, of which 45 were up-regulated and 18 were down-regulatedConclusion: Our preliminary study showed that G0S2 overexpression in the liver led to specific protein spectrum changes, with 63 differentially expressed proteins involved in the regulation of apoptosis, elucidating an important relationship between G0S2 and apoptosis. This finding indicated that G0S2 may involve in cancer, neurodegenerative diseases and numerous metabolic diseases an apoptotic factor, providing new insights into the mechanisms of disease development.

Difenoconazole (DCZ) is a widely used triazole fungicide and negatively affects aquatic organisms and human health. However, data on the reproductive toxicity of DCZ are lacking. In this study, we used zebrafish (from 2–4 h postfertilization (hpf) to adulthood) as a model to evaluate whether DCZ at environmentally relevant concentrations (0.1, 1.0, and 10.0 µg/L) induce reproductive toxicity. Following exposure to DCZ, egg production and fertilization rate were reduced at 1.0 and 10.0 µg/L. A significant decrease in the proportion of mature gametes (late vitellogenic oocytes and spermatozoa) was also observed at 10.0 µg/L. The concentrations of 17β-Estradiol (E2), testosterone (T), and vitellogenin (VTG) were disrupted in females and males at 1.0 and 10.0 µg/L. Exposure to 10.0 µg/L DCZ significantly inhibited the contact time between female fish and male fish, and it was mainly achieved by affecting male fish. The transcription of genes involved in the hypothalamus-pituitary-gonad-liver (HPGL) axis was significantly changed after treatment with DCZ. Overall, these data showed that the endocrine disrupting effect of DCZ on the zebrafish HPGL axis inhibited the formation of gametes and disrupted reproductive behavior, thereby reducing fertility.

Resistance to chemotherapy drugs makes ovarian cancer (OC) difficult to treat and ultimately kills patients. Long non-coding RNAs are closely related to carboplatin resistance in OC. In present study, we explored the role of lncRNA X-inactive specific transcript (XIST) on carboplatin resistance in OC.Cell viability, proliferation, and apoptosis were assessed through 2,5-diphenyl-2H-tetrazolium bromide, colony formation, and flow cytometry assays, respectively. Microtubule-associated protein 1A/1B-light chain 3 expression was evaluated by immunofluorescence assay to analyze the cell autophagy. The interaction of XIST/miR-506-3p or miR-506-3p/forkhead box protein P1 (FOXP1) was analyzed using RNA immunoprecipitation (RIP) and dual-luciferases reporter assays. The function of XIST/miR-506-3p/FOXP1 axis in vivo was further confirmed by tumor xenograft study and immunohistochemistry.The expression of XIST and FOXP1 increased while miR-506-3p decreased in OC and carboplatin resistance cells. XIST silencing repressed the proliferative and autophagic capacities of carboplatin resistance cells while promoted the apoptosis. XIST overexpression led to the opposite results. XIST targeted miR-506-3p and downregulated its expression. MiR-506-3p inhibition facilitated the proliferative and autophagic capacities while suppressed the apoptosis of cells, XIST knockdown reversed these effects. MiR-506-3p bound to FOXP1. XIST knockdown or miR-506-3p overexpression reversed the increase of cell proliferative and autophagic abilities and the decrease of apoptosis rate induced by FOXP1 overexpression. XIST affected autophagy and carboplatin resistance in vivo via regulating the miR-506-3p/FOXP1 axis.XIST knockdown inhibited autophagy and carboplatin resistance of OC through FOXP1/protein kinase B (AKT)/mammalian target of rapamycin pathway by targeting miR-506-3p.

Development of rapid and accurate detection of miRNAs in complex samples is of great significance for potential early diagnosis of disease. Herein, we report a magnetic relaxation switching (MRS)-based strategy for direct detection of miRNAs in complex samples via the assistance of signal amplification system of CRISPR/Cas13a which has the ability to specifically recognize target RNA. In the designed strategy, 30 nm-magnetic nanoparticles (MB

Multiple sclerosis disproportionally affects women. The present study was undertaken to determine whether NFAT5 contributed to the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and if it did, whether the impact was sex associated. NFAT5 haplodeficiency reduced the disease severity only in female mice. This effect was associated with significant increases in frequency of T regulatory (Treg) cells in the CNS (from 1.45 ± 0.39% to 3.73 ± 0.94%) and spleen from (0.31 ± 0.06% to 0.94 ± 0.29%) without significantly affecting the CNS CD4

To cope with the challenge of successful edge offloading brought by the mobility of mobile devices in intelligent factories, this paper studies the optimization problem of the edge offloading strategy of mobile devices based on mobility. Considering the decision task flow executed by priority, the unique offloading mode of a single task, the communication range of the edge server, and the delay constraint of the offloading of a single task, appropriate computing resources are selected according to the real-time location of the mobile device to offload the computing task. Based on the edge computing architecture of an intelligent factory, this paper puts forward five different computation offloading methods. From a global perspective, the energy consumption and delay of tasks offloading in local, edge, cloud center, local-edge collaboration, and local-edge-cloud collaboration are considered. In this paper, the algorithm based on the genetic algorithm and particle swarm optimization is used to design and obtain the decision task flow offloading strategy with the lowest energy consumption and delay. Simulation results show that the proposed algorithm can reduce the computation offloading energy consumption and delay of mobile devices.

In viticulture, grafting has been practiced widely and influences grape development as well as berry and wine quality. However, there is limited understanding of the effects of rootstocks on grape phenolic compounds, which are located primarily in the berry skin and contribute to certain sensory attributes of wine. In this study, scion–rootstock interactions were investigated at the green-berry stage and the veraison stage when grapevines were hetero-grafted with three commonly used rootstock genotypes (5BB, 101-14MG, and SO4). Physiological investigations showed that hetero-grafts, especially CS/5BB, contained higher concentrations of total proanthocyanidins (PAs) and various PA components in berry skins compared with the auto-grafted grapevines. Further metabolomics analysis identified 105 differentially accumulated flavonoid compounds, the majority of which, including anthocyanins, PAs, and flavonols, were significantly increased in the berry skins of hetero-grafted grapevines compared with auto-grafted controls. In addition, transcriptomic analysis of the same samples identified several thousand differentially expressed genes between hetero-grafted and auto-grafted vines. The three rootstocks not only increased the transcript levels of stilbene, anthocyanin, PA, and flavonol synthesis genes but also affected the expression of numerous transcription factor genes. Taken together, our results suggest that hetero-grafting can promote phenolic compound accumulation in grape berry skin during development. These findings provide new insights for improving the application value of grafting by enhancing the accumulation of nutritious phenolic components in grape.

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here we report the structure and function PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer and each protomer has 14 transmembrane segments folded into a substrate-binding domain (core domain) and an interface domain (gate domain) A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines, and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

Developmental exposure to bisphenol A (BPA), an endocrine-disrupting contaminant, impairs cognitive function in both animals and humans. However, whether BPA affects the development of primary sensory systems, which are the first to mature in the cortex, remains largely unclear. Using the rat as a model, we aimed to record the physiological and structural changes in the primary auditory cortex (A1) following lactational BPA exposure and their possible effects on behavioral outcomes. We found that BPA-exposed rats showed significant behavioral impairments when performing a sound temporal rate discrimination test. A significant alteration in spectral and temporal processing was also recorded in their A1, manifested as degraded frequency selectivity and diminished stimulus rate-following by neurons. These post-exposure effects were accompanied by changes in the density and maturity of dendritic spines in A1. Our findings demonstrated developmental impacts of BPA on auditory cortical processing and auditory-related discrimination, particularly in the temporal domain. Thus, the health implications for humans associated with early exposure to endocrine disruptors such as BPA merit more careful examination.

Mutations causative of neurological and neurodegenerative disease can occur in coding regions that specify protein domains of low sequence complexity. These autosomal dominant mutations can be idiosyncratic in their recurrent appearance at the same amino acid. Here we report studies of recurrent mutations in proline residues located within low complexity (LC) domains associated with the neurofilament light chain protein, the microtubule-associated tau protein, and the heterogeneous nuclear RNPA2 protein. All such mutations manifest their effects by directing formation of variant proteins endowed with the addition of a single, main chain hydrogen bond specified by the variant amino acid replacing proline. Here we show that methylation of the peptide backbone nitrogen atom associated with these variant amino acids eliminates the aberrant hydrogen bond and restores normal protein function.

TAR binding protein 43 (TDP-43) is an RNA binding protein that assists in the maturation, export and sub-cellular localization of mRNA. The carboxyl terminal 153 residues of TDP-43 are of low sequence complexity and allow for self-association of the protein in a manner leading to its phase separation from an aqueous environment. These interactions assist TDP-43 in forming cytoplasmic RNA granules involved in the transport of mRNA for localized translation. Self-association of the TDP-43 low complexity (LC) domain is facilitated by a region of twenty five residues that are of extreme evolutionary conservation. The molecular basis for self-adherence of the protein through this region has been illuminated by a combination of structural and biochemical studies, allowing definition of a morphologically specific cross-β structure predicted to be weakly assembled by main chain hydrogen bonds. In this study we have investigated the importance of individual, Pauling hydrogen bonds hypothesized to facilitate self-adherence of the TDP-43 LC domain.

Non-Hermitian systems with parity-time (PT) symmetry reveal rich physics beyond the Hermitian regime. As the counterpart of conventional PT symmetry, anti-parity-time (APT) symmetry may lead to new insights and applications. Complementary to PT-symmetric systems, non-reciprocal and chiral mode switching for symmetry-broken modes have been reported in optics with an exceptional point dynamically encircled in the parameter space of an APT-symmetric system. However, it has remained an open question whether and how the APT-symmetry-induced chiral mode transfer could be realized in mechanical systems. This paper investigates the implementation of APT symmetry in a three-element mass–spring system. The dynamic encircling of an APT-symmetric exceptional point has been implemented using dynamic-modulation mechanisms with time-driven stiffness. It is found that the dynamic encircling of an exceptional point in an APT-symmetric system with the starting point near the symmetry-broken phase leads to chiral mode switching. These findings may provide new opportunities for unprecedented wave manipulation in mechanical systems.

Hepatic fibrosis is an important early stage in the evolution of liver cirrhosis, and specific medicine and therapeutic measures are unavailable to date. Hepatic stellate cells (HSCs) are the main cells involved in the formation of hepatic fibrosis, and induction of the apoptosis of HSCs is an important strategy for the treatment of hepatic fibrosis. Diallyl trisulfide (DATS) is a natural product and is the main active ingredient in garlic. However, the exact molecular mechanisms underlying HSC apoptosis induced by DATS are not well understood. This study aimed to analyze the efficiency and mechanism of DATS in hepatic fibrosis. Different concentrations (25, 50, 100, and 200 μM) of DATS were used to treat HSCs. Changes in cell morphology and formation of apoptotic bodies were observed under an inverted microscope and an electric microscope. Bcl-2 signaling involving Bax, Caspase-3, Caspase-6, Caspase-8, Caspase-9, p53, Apaf-1, and Cyto-c in fibrosis were examined, which is a critical step in the evaluation of antihepatic fibrosis agents. We also evaluated the effect of DATS on the cellular morphology of HSCs and apoptosis-related factors under different Bcl-2 expression states. Our results suggest that DATS regulates hepatic fibrosis by blocking the Bcl-2 signaling pathway and upregulating the Bax/Bcl-2 ratio.

Background: Endometriosis (EMs) is the most common gynaecological disorder with its etiology and/or pathophysiology remains enigmatic. Recent studies showed that extracellular vesicles (EVs), exosomes in particular, play a critical role in developing various clinical disorders. However, the implication of exosomes in endometriosis progression has not been well elucidated.Method: The ectopic stromal cellular exosomes (eEVs) were assessed by transwell assay, scratch tests, tube formation assay, western blot, and qRT-PCR analysis. Protein expression profiles of exosomes in endometrial tissue and vaginal discharge collected from patients with EMS and healthy donors were analysed by Mass spectrometry. siRNA interference technology was used to inhibit the expression of exosomal protein for the functional analysis in in-vivo. Finally, in-vitro experiments were performed to validate the results that we observed in EMs mouse model.Results:In vitro, we discovered that eEVs improved NSC migratory potential by upregulating MMP9 expression and activity. eEVs also aided angiogenesis and elevated the expression of inflammatory cytokines in ovarian epithelial cells, according to our findings. Moesin (MSN) levels in ESC exosomes were substantially greater than in NSC exosomes (1.22e8±5.58e6 vs. 6.605e7±4.574e6, LFQ intensity), as shown by protein mass spectrometry and bioinformatics analysis. In ectopic stromal cells, ERa receptors stimulated the RhoA/Rock-2/MSN pathway. We discovered that downregulating exosomal moesin reduced NSC migration (about 3-fold change) and MMP9 expression (about 2-fold change). On the other hand, Exomsni inhibited angiogenesis and inflammatory cytokine release. In vivo the result of immunohistochemistry and immunofluorescence demonstrated that exosomal MSN substantially modified the expression of MM9, VEGFR and p-VEGFR in polyclonal lesions. In addition, we discovered an elevation in the expression of proinflammatory factors in the surrounding tissue.Conclusion: Exosomal MSN derived from ectopic stromal cells can contribute to endometriosis progression by mediating the construction of a “migration-vascularization-inflammation” loop in the ectopic environment.

The mechanisms by which lower limb ischemia/reperfusion induces acute kidney injury (AKI) remain largely uncharacterized. We hypothesized that tourniquet-induced lower limb ischemia/reperfusion (TILLIR) would inhibit mitochondrial function in the renal cortex. We used a murine model to show that TILLIR of the high thigh regions inflicted time-dependent AKI as determined by renal function and histology. This effect was associated with decreased activities of mitochondrial complexes I, II, V and citrate synthase in the kidney cortex. Moreover, TILLIR reduced mRNA levels of a master regulator of mitochondrial biogenesis PGC-1α, and its downstream genes NDUFS1 and ATP5o in the renal cortex. TILLIR also increased serum corticosterone concentrations. TILLIR did not significantly affect protein levels of the critical regulators of mitophagy PINK1 and PARK2, mitochondrial transport proteins Tom20 and Tom70, or heat-shock protein 27. TILLIR had no significant effect on mitochondrial oxidative stress as determined by mitochondrial ability to generate reactive oxygen species, protein carbonylation, or protein levels of MnSOD and peroxiredoxin1. However, TILLIR inhibited classic autophagic flux by increasing p62 protein abundance and preventing the conversion of LC3-I to LC3-II. TILLIR increased phosphorylation of cytosolic and mitochondrial ERK1/2 and mitochondrial AKT1, as well as mitochondrial SGK1 activity. In conclusion, lower limb ischemia/reperfusion induces distal AKI by inhibiting mitochondrial function through reducing mitochondrial biogenesis. This AKI occurs without significantly affecting PINK1-PARK2-mediated mitophagy or mitochondrial oxidative stress in the kidney cortex.