Protein Activity Research Articles

Bacterial recognition and inhibition activities of an LRR-only protein in the Chinese mitten crab, Eriocheir sinensis

LRR-only protein (LRRop) is an important class of immune molecules that function as pattern recognition receptor in invertebrates, however, the bacterial inhibitory activity of this proteins remain largely unknown. Herein, a novel LRRop was cloned from Eriocheir sinensis and named as EsLRRop2. The EsLRRop2 consists of six LRR motifs and formed a horseshoe shape three-dimension structure. EsLRRop2 was mainly expressed in intestine and hepatopancreas. The transcripts of EsLRRop2 in the intestine and hepatopancreas were induced by Vibrio parahaemolyticus and Staphylococcus aureus, and displayed similar transcriptional profiles. The expression levels of EsLRRop2 responded more rapidly and highly to V. parahaemolyticus than S. aureus in the intestine and hepatopancreas. Although the basal expression level of EsLRRop2 in hemocytes was relatively low, its transcripts in hemocytes were significantly induced by V. parahaemolyticus and S. aureus. The recombinant proteins of EsLRRop2 (rEsLRRop2) displayed a wide range of binding spectrum against vibrios, including V. parahaemolyticus, V. alginolyticus, and V. harveryi. The rEsLRRop2 showed dose- and time-dependent inhibitory activity against V. parahaemolyticus and S. aureus, and it could agglutinate the two bacteria. Furthermore, the inhibitory activities of rEsLRRop2 against V. parahaemolyticus, V. alginolyticus, V. harveryi and S. aureus was slightly affected by pH and salinity, and the rEsLRRop2 displayed the strongest inhibitory activity against all the three vibrios when the salinity was 20‰ and pH was 8.0. Collectively, these results elucidate the bacterial binding and inhibitory activities of EsLRRop2, and provide theoretical foundations for the application of rEsLRRop2 in prevention and control of vibrio diseases in aquaculture.

Synthesis and characterization of 64Cu-labeled Geldanamycin derivative for imaging HSP90 expression in breast cancer

Heat shock protein 90 (HSP90) plays a crucial role in cancer cell growth and metastasis by stabilizing overexpressed signaling proteins. Inhibiting HSP90 has emerged as a promising anti-cancer strategy. In this study, we aimed to develop and characterize a HSP90-targeted molecular imaging probe, [64Cu]Cu-DOTA-BDA-GM, based on a specific HSP90 inhibitor, geldanamycin (GM), for PET imaging of cancers. GM is modified at the C-17 position with 1,4-butane-diamine (BDA) and linked to 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for 64Cu radiolabeling. We evaluated the probe's specific binding to HSP90-expressing cells using Chinese hamster ovary (CHO) cells and breast cancer cells including MDA-MB-231, MDA-MB-435S, MCF7, and KR-BR-3 cell lines. A competition study with non-radioactive GM-BDA yielded an IC50 value of 1.35 ± 0.14 nM, underscoring the probe's affinity for HSP90. In xenograft models of MDA-MB-231 breast cancer, [64Cu]Cu-DOTA-BDA-GM showcased targeted tumor localization, with significant radioactivity observed up to 18 h post-injection. Blocking studies using unlabeled GM-BDA and treatment with the anticancer drug Vorinostat (SAHA), which can affect the expression and activity of numerous proteins, such as HSPs, confirmed the specificity and sensitivity of the probe in cancer targeting. Additionally, PET/CT imaging in a lung metastasis mouse model revealed increased lung uptake of [64Cu]Cu-DOTA-BDA-GM in metastatic sites, significantly higher than in non-metastatic lungs, illustrating the probe's ability to detect metastatic breast cancer. In conclusion, [64Cu]Cu-DOTA-BDA-GM represents a sensitive and specific approach for identifying HSP90 expression in breast cancer and metastases, offering promising implications for clinical diagnosis and monitoring.

Efficacy and Safety of Phosphatidylinositol 3-kinase Inhibitors for Patients with Breast Cancer: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

Phosphatidylinositol 3-kinase (PI3K) inhibitors belong to the class of drugs that inhibit the activity of the PI3K protein, which is commonly overexpressed in breast cancer cells. However, there is a need to summarize the evidence to provide conclusive advice on the benefit of PI3K inhibitors in breast cancer patients. Therefore, this review assessed the effectiveness and safety of the PI3K inhibitors amongst breast cancer patients. Searches were made in PubMed Central, EMBASE, MEDLINE, SCOPUS, CENTRAL, WHO trial registry and Clinicaltrials.gov up to December 2022. Meta-analysis was executed using the random-effects model. Pooled hazard ratio (HR)/risk ratio (RR) was reported with 95% confidence intervals (CIs). In total, 13 studies were included in the analysis. Most were multi-country studies and had a higher risk of bias. Regarding the efficacy parameters, pooled HR for progression-free survival was 0.79 (95%CI: 0.67-0.92), pooled RR for complete response was 1.54 [95%CI: 1.14 to 2.09], partial response was 1.18 [95%CI: 0.87-1.61], overall response was 1.20 [95%CI: 0.93-1.56], stable disease was 1.09 [95%CI: 0.78-1.53], progressive disease was 0.80 [95%CI: 0.74 to 0.87], and clinical benefit was 1.08 [95%CI: 0.80-1.49]. For safety parameters, pooled RR for hyperglycemia was 4.57 [95%CI: 3.15-6.62], and gastrointestinal toxicity was 1.82 [95%CI: 1.56 to 2.14]. PI3K inhibitors had better efficacy than the present standard of concern for patients with breast cancer, especially among patients with PIK3CA mutations. Hence, clinicians and oncologists can provide this drug for the target population with extra caution for diabetes patients.

Discovery of natural rosin-based preservative candidates to control Phytophthora capsici for postharvest disease management of solanaceae vegetables

To mitigate the postharvest harm of pathogens on solanaceous vegetables, a series of natural rosin-1,2,4-triazole derivatives (totally 24) have been designed and developed. In vivo fungicidal activity evaluation showed that Co. 5 f could inhibit the growth of Phytophthora capsici (EC50 = 0.22 mg/L). In vivo activity suggested that Co. 5 f presented certain protective effect on bell pepper (64.93 % – 32.68 %) and tomato (91.93 % – 67.85 %) fruit. Additionally, Co. 5 f prevented declines in both weight and ascorbic acid levels and reduced the malondialdehyde content of bell peppers and tomato, suggesting its efficacy in vegetable preservation during storage. The physiological research revealed that Co. 5 f disrupted the mycelial cell membrane, accelerated the accumulation of reactive oxygen species, and decreased the enzyme activity of peroxidase (POD) and catalase (CAT) protein activities. Subsequently, quantum chemical calculations and molecular docking successfully predicted the active site of Co. 5 f. Metabolomics was then employed to elucidate the mechanism of physiological disorders, suggesting that differential metabolites were primarily enriched in the biosynthesis of amino acid and secondary metabolites. Transcriptomics was also utilized to further uncover the mechanism of mycelial metabolic disorders, revealing that differential genes were primarily enriched in the biosynthesis of secondary metabolites and amino acid. Additionally, the integration of physiology, metabolomics, transcriptomics, and computer-aided drug design unveiled the action mechanism of Co. 5 f against P. capsici, indicating that the regulation of POD and CAT biosynthesis involved six core genes, while the regulation of ARG, HIS, and TRP biosynthesis involved four core genes. Finally, the expression of these ten core genes was validated using quantitative real-time polymerase chain reaction (qRT-PCR). In summary, the above research integrated physiology, metabolomics, transcriptomics and computer-aided drug design to reveal the action mechanism of Co. 5 f, providing a certain theoretical support for the postharvest disease management of bell pepper and tomato fruit.

Design of scalable manufacturing process for the production of biodegradable polymeric microneedles for protein transdermal drug delivery

Introduction. Dissolving polymeric microneedles are attractive drug delivery system especially for vaccine delivery. Still there are a lot of obstacles in developing scalable manufacturing process of them.Aim. To develop a scalable manufacturing process for producing polymeric dissolving microneedles, which can enable keeping protein activity during manufacturing process.Materials and methods. Microneedles were produced from aqua solution of 20 % w.v. pullulan and 3 % w.v. polyvinyl alcohol by casting in hollow negative polyethylene terephthalate mold. Human serum albumin was chosen as a model protein for this investigation.Results and discussion. There were chosen the mode of mold filling and microneedle drying process, which can guarantee keeping of protein activity during manufacturing process.Conclusion. The designed technology can be easily scaled up and used for producing vaccine drug delivery systems, because it doesn’t contain any restraining processes.

Knockdown of SMYD3 by RNA Interference Regulates the Expression of Autophagy-Related Proteins and Inhibits Bone Formation in Fluoride-Exposed Osteoblasts.

This study aimed to explore the role of histone methyltransferase SET and MYND domain containing 3 (SMYD3) in bone metabolism of osteoblasts exposed to fluoride. The levels of urine fluoride, BALP, and OC and the mRNA expression of SMYD3 were determined in patients with skeletal fluorosis and non-fluoride-exposed people on informed consent. The expression of SMYD3 protein, OC contents, and BALP activities were detected in human osteoblast-like MG63 cells and rat primary osteoblasts treated with sodium fluoride (NaF) for 48 h. The autophagosomes were observed by transmission electron microscopy. Then, we knocked down SMYD3 to confirm whether it was involved in the regulation of bone formation and related to autophagy and Wnt/β-catenin pathway. We observed that OC and BALP levels in patients with skeletal fluorosis significantly increased, while the mRNA expression of SMYD3 significantly decreased in the skeletal fluorosis groups. In vitro, the OC contents, BALP activities, and expression of SMYD3 significantly increased, and many autophagosomes were observed in NaF treated osteoblasts. The downregulation of SMYD3 significantly inhibited OC contents, BALP activities, and expression of autophagy-related proteins, but with no significant changes in the Wnt/β-catenin pathway. Our results demonstrated that fluoride exposure with coal-burning pollution caused orthopedic injuries and abnormalities in the levels of OC and BALP and hindered normal bone metabolism. Silencing the SMYD3 gene could significantly reduce OC and BALP levels via inhibiting the increase in autophagy induced by fluoride.

Serum Leucine Aminopeptidase Activity Patterns Across Various Disease States: Potential Implications for Bleeding and Thrombosis Risk.

Disruptions in the pathways for activating and deactivating proteases in the bloodstream can lead to thrombosis and bleeding issues. Leucine aminopeptidases (LAPs), which are exopeptidases essential for regulating protein and peptide activities, are recognized as clinical biomarkers for liver diseases. However, the relationship between serum LAP activity and the risks of bleeding or thrombosis, as well as the identification of the specific tissues or organs that control LAP levels, is not well understood. We performed a retrospective study to evaluate serum LAP activities in 149,360 patients with 47 different diseases and 9,449 healthy individuals. The analysis was conducted using SPSS V2.6, RStudio V.1.3.1073, and libraries in Python 3.8. Our research revealed that 21 of the 47 diseases studied showed increased median serum LAP activities, while 26 diseases were associated with significantly lower activities, especially those related to thrombosis. Furthermore, most diseases were found to have an increased risk of bleeding and thrombosis, indicated by higher Q25 and lower Q75 LAP activities compared to the control group. Receiver operating characteristic curve analysis confirmed the effectiveness of LAP activities as biomarkers for specific conditions like hepatic encephalopathy, liver cancer, pancreatitis, and pancreatic cancer. Diseases were categorized into clusters with similar bleeding or thrombotic tendencies through principal component analysis. This study highlighted regulatory influence of the liver and pancreas on LAP levels. The established link between serum LAP concentrations and the risk of bleeding or thrombosis paved the way for the development of diagnostic and preventative approaches for various medical conditions.

Machine Learning-Driven Biomarker Discovery for Skeletal Complications in Type 1 Gaucher Disease Patients

Type 1 Gaucher disease (GD1) is a rare, autosomal recessive disorder caused by glucocerebrosidase deficiency. Skeletal manifestations represent one of the most debilitating and potentially irreversible complications of GD1. Although imaging studies are the gold standard, early diagnostic/prognostic tools, such as molecular biomarkers, are needed for the rapid management of skeletal complications. This study aimed to identify potential protein biomarkers capable of predicting the early diagnosis of bone skeletal complications in GD1 patients using artificial intelligence. An in silico study was performed using the novel Therapeutic Performance Mapping System methodology to construct mathematical models of GD1-associated complications at the protein level. Pathophysiological characterization was performed before modeling, and a data science strategy was applied to the predicted protein activity for each protein in the models to identify classifiers. Statistical criteria were used to prioritize the most promising candidates, and 18 candidates were identified. Among them, PDGFB, IL1R2, PTH and CCL3 (MIP-1α) were highlighted due to their ease of measurement in blood. This study proposes a validated novel tool to discover new protein biomarkers to support clinician decision-making in an area where medical needs have not yet been met. However, confirming the results using in vitro and/or in vivo studies is necessary.

Cryo-EM structure of 1-deoxy-D-xylulose 5-phosphate synthase DXPS from Plasmodium falciparum reveals a distinct N-terminal domain

Plasmodium falciparum is the main causative agent of malaria, a deadly disease that mainly affects children under five years old. Artemisinin-based combination therapies have been pivotal in controlling the disease, but resistance has arisen in various regions, increasing the risk of treatment failure. The non-mevalonate pathway is essential for the isoprenoid synthesis in Plasmodium and provides several under-explored targets to be used in the discovery of new antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of the pathway. Despite its importance, there are no structures available for any Plasmodium spp., due to the complex sequence which contains large regions of high disorder, making crystallisation a difficult task. In this manuscript, we use cryo-electron microscopy to solve the P. falciparum DXPS structure at a final resolution of 2.42 Å. Overall, the structure resembles other DXPS enzymes but includes a distinct N-terminal domain exclusive to the Plasmodium genus. Mutational studies show that destabilization of the cap domain interface negatively impacts protein stability and activity. Additionally, a density for the co-factor thiamine diphosphate is found in the active site. Our work highlights the potential of cryo-EM to obtain structures of P. falciparum proteins that are unfeasible by means of crystallography.

Betaine and the probiotic, Lactobacillus rhamnosus in the diet of the Common carp, Cyprinus carpio: Effects on growth, digestive enzyme activities, antioxidant system, humoral and mucosal immunity and resistance to Streptococcus iniae

The probiotic, Lactobacillus rhamnosus (LAC-R) and Betaine (BT) alone and combined were used in the diet of the common carp, Cyprinus carpio to evaluate their potentials in improving growth, immunity and disease resistance. Seven experimental treatments (LAC-R: 1 × 108 CFU/g LAC-R; BT1: 0.5 % BT; BT2: 1 % BT; BT3: 2 % BT; LAC-R + BT1; LAC-R + BT2; LAC-P + BT3) and one control group (control: non-supplemented fish) were designed in three replicates and fish (13.3 ± 1.5 g) fed with the test diets for two months. After the feeding experiment, fish were exposed to Streptococcus iniae for 14 days to evaluate the fish disease resistance. The fish supplemented with BT and LAC-R alone or combined exhibited better growth compared to control (P<0.01), however, fish of LAC-R + BT had the highest growth performance (P<0.01). Digestive enzyme activities increased in response to LAC-R, BT and their combinations, in which the highest activities were for LAC-R + BT2 and LAC-R + BT3 (P<0.01). The lactic acid bacteria (LAB) populations in intestine increased after the probiotic supplementation (P<0.01), while the results were non-significant for total bacteria count (P>0.01). The total antioxidant capacity (T-AOC), antioxidant enzymes [serum catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx)] and immune components [lysozyme, total immunoglobulin (Ig), myeloperoxidase (MPO), alternative complement activity (ACH50), Nitroblue tetrazolium (NBT) and total protein in blood and total Ig, lysozyme, alkaline phosphatase (ALP) and protease in mucus] were mostly enhanced by LAC-R, BT2, BT3 and their combinations (P<0.01). The oxidative stress was ameliorated by LAC-R, BT and their combinations, as the levels of malondialdehyde (MDA) were lower in these treatments (P<0.01). Fish supplemented with LAC-R, BT and LAC-R + BT had lower mortality rate compared to control after challenge with S. Iniae (P<0.01). In conclusion, dietary LAC-R and BT improved growth and immune performance in the fish. Also, the best performance was found for fish supplemented with a combination of LAC-R and 1–2 % BT.

Selenium nanoparticles alleviates cadmium induced hepatotoxicity by inhibiting ferroptosis and oxidative stress in vivo and in vitro

Cadmium (Cd) is an important environmental toxicant that could cause serious damage to various organs including severe hepatotoxicity in intoxicated animals. Selenium has been reported to possess the protective effects against Cd toxicity, but the specific mechanism is still unclear. The purpose of this study was to explore the effects and mechanism of chitosan coated selenium nanoparticles (CS-SeNPs) against Cd-induced hepatotoxicity in animal and cellular models. ICR mice and rat hepatocyte BRL-3A cells were exposed to cadmium chloride (CdCl2) to evaluate the therapeutic efficiency of CS-SeNPs. Analysis of histopathological images, mitochondrial membrane potential (MMP) and ultramicrostructure, serum liver enzyme activities, ferroptosis-related indicators contents, and further molecular biology experiments were performed to investigate the underlying mechanisms. In vivo experiment results showed that CdCl2 caused significant pathological damage involving significant increase of liver index, contents of tissue MDA and serum ALT and AST, and significant decrease of serum GSH-Px activity. Moreover, CdCl2 exposure upregulated ACSL4 and HO-1 protein levels, downregulated GPX4, TfR1, ferritin protein levels in the liver. Notably, CS-SeNPs increased the expression level of GPX4 and ameliorated CdCl2-induced changes in above-mentioned indicators. In vitro experimental results showed that treatment with CS-SeNPs significantly elevated GSH-Px activity and GPX4 protein level, reversed CdCl2-induced expression of several ferroptosis-related proteins TfR1, FTH1 and HO-1, and repressed ROS production and increased MMP of the cells exposed to CdCl2. Our research indicated that CdCl2 induced hepatocyte injury by inducing ferroptosis, while CS-SeNPs can inhibit ferroptosis and reduce the degree of hepatocyte injury. This study is of great significance for further revealing the mechanism of Cd hepatotoxicity and expanding the clinical application of SeNPs.

Salidroside exerts anti-tumor effects in ovarian cancer by inhibiting STAT3/c-Myc pathway-mediated glycolysis.

Salidroside (SAL) is a bioactive substance extracted from the traditional Chinese medicine Rhodiola rosea, which exhibits multiple pharmacological effects, such as anti-inflammatory, antioxidant, and anti-tumor properties. Currently, the effects of SAL on the malignant progression of ovarian cancer (OC) and its specific mechanism of action are not clear. Cell Counting Kit 8 (CCK-8), clone formation, Hoechst 33258 staining, flow cytometry, transwell, western blotting and immunofluorescence assays were performed to determine the impacts of SAL on the biological properties of OC cells (CAOV3 and SKOV3) and human normal ovarian epithelial cells (IOSE80). The binding activity of SAL and proteins was evaluated. Glucose consumption, lactate and ATP production, extracellular acidification rate (ECAR) and related proteins were measured to assess glycolysis. Animal models were established to evaluate the impact of SAL treatment in vivo and the expression levels of STAT3/c-Myc pathway-related proteins were determined to explore the relationship between SAL and OC. The results showed that SAL reduced the viability, clone formation, migration and invasion ability of CAOV3 and SKOV3 cells, and induced apoptosis. SAL inhibited epithelial-mesenchymal transition (EMT) and decreased glucose consumption, lactate and ATP production and ECAR. SAL exhibited good binding activity with STAT3 and c-Myc and reduced the expression levels of STAT3/c-Myc pathway and glycolysis-related proteins in vitro and in vivo. In conclusion, SAL exerted anti-tumor effects by interfering with the malignant biological progression of OC cells by inhibiting STAT3/c-Myc pathway-mediated glycolysis.

Thermal stability of bivalent cation/phosphoinositide domains in model membranes

As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg2+ ions. While PI(4)P shows in the presence of Ca2+ no clustering, PI(4,5)P2 forms with Ca2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P3 with Ca2+ is less than what was observed for PI(4,5)P2, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg2+ or Ca2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca2+ with PI(4,5)P2. Also, in the presence of cholesterol, the interaction of Mg2+ with PI(4,5)P2 remains weak. PI(3,4,5)P3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca2+ and Mg2+ was not influenced by the presence of cholesterol.

Hansenula polymorpha methanol metabolism genes enhance recombinant protein production in Komagataella phaffi

Recombinant protein production in Komagataella phaffi (K. phaffi), a widely utilized host organism, can be optimized by enhancing the metabolic flux in the central carbon metabolism pathways. The methanol utilization pathway (MUT) during methanol-based growth plays a crucial role in providing precursors and energy for cell growth and development. This study investigated the impact of boosting the methanol dissimilation pathway, a branch of MUT that plays a vital role in detoxifying formaldehyde and providing energy in the form of NADH, in K. phaffi. This was achieved by integrating two orthologous genes from Hansenula polymorpha into the K. phaffi genome: formaldehyde dehydrogenase (HpFLD) and formate dehydrogenase (HpFMDH). The HpFLD and HpFMDH genes were isolated from the Hansenula polymorpha genome and inserted under the regulation of the pAOX1 promoter in the genome of recombinant K. phaffi that already contained a single copy of model protein genes (eGFP or EGII). The expression levels of these model proteins were assessed through protein activity assays and gene expression analysis. The findings revealed that while both orthologous genes positively influenced model protein production, HpFMDH exhibited a more pronounced upregulation in expression compared to HpFLD. Co-expression of both orthologous genes demonstrated synergistic effects, resulting in approximately a twofold increase in the levels of the model proteins detected. This study provides valuable insights into enhancing the production capacity of recombinant proteins in K. phaffi.Graphical abstract

Knockdown of LRCH4 Remodels Tumor Microenvironment Through Inhibiting YAP and TGF-β/Smad Signaling Pathway in Colorectal Cancer.

Colorectal cancer is one of the most common gastrointestinal malignancies worldwide. LRCH4 is the top 1 gene associated with an unfavorable prognosis in colorectal cancer. Here, we reported that the knockdown of LRCH4 inhibited the proliferation, migration and invasion in HT29 cells. The activity of Yes-Associated Protein (YAP), a transcription factor in the Hppo-YAP signaling pathway, was significantly inhibited by LRCH4-siRNA. LRCH4 knockdown also reversed the EMT and regulated the expression of extracellular matrix (ECM) protein, Fibronectin and Collagen IV in HT29 cells. In addition, the TGF-β/Smad signaling pathway, as the downstream pathway of Yap, was also inhibited by LRCH4 knockdown. Knockdown of LRCH4 involved in the regulation of ECM and EMT and inhibited YAP and the TGF-β/Smad signaling pathway in colorectal cancer cells. Our study provided a mechanism of LRCH4 on colorectal cancer cells, and a new potential target for clinical tumor treatment.

Growth performance, mucosal immunity and disease resistance in goldfish (Carassius auratus) orally administered with Escherichia coli Strain Nissle 1917.

The current research aimed to shed light on the efficacy of Escherichia coli strain Nissle 1917 (EcN) on goldfish (……) growth, gut immunity, morphology, bacterial nutritional enzyme activity and resistance to Aeromonas hydrophila infection. Fish fed with EcN at 106, 107 and 108CFU/g feed for 80days showed an enhancement in growth better than control fish. The gut innate immunity in terms of lysozyme activity, immunoglobulin and total protein levels was increased in the treatment fish with the best result being observed in fish fed EcN at 108CFU/ g. In addition, an increase was noted in the upregulation of immune-relevant genes, namely lysozyme, interleukin-1β, inducible nitric oxide synthase and tumor necrosis factor α of fish intestine. A marked surge in the number of proteolytic and heterotrophic bacteria was noted in the gut of fish nourished with the probiotic. Histological studies exhibited an improvement in the intestinal absorption surface area, intraepithelial lymphocyte count and goblet cell density. Significantly higher survival rate was obtained in fish fed EcN at 108CFU/g compared with the fish fed with the basal diet. These data exhibited the beneficial effect of EcN on goldfish growth, digestive enzymes, intestine heterotrophic bacteria and resistance against Aeromonas hydrophila challenge. This study confirmed the favorable outcomes resulting from the administration of EcN at108 CFU/g.

Cell size regulates human endoderm specification through actomyosin-dependent AMOT-YAP signaling

Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.

Signalling of the neuropeptide calcitonin gene-related peptide (CGRP) through RAMP1 promotes liver fibrosis via TGFβ1/Smad2 and YAP pathways

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFβ1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFβ1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFβ1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFβ1/Smad2 and YAP activity.

Emerging role of sirtuins in non‑small cell lung cancer (Review).

Non‑small cell lung cancer (NSCLC) is a highly prevalent lung malignancy characterized by insidious onset, rapid progression and advanced stage at the time of diagnosis, making radical surgery impossible. Sirtuin (SIRT) is a histone deacetylase that relies on NAD+ for its function, regulating the aging process through modifications in protein activity and stability. It is intricately linked to various processes, including glycolipid metabolism, inflammation, lifespan regulation, tumor formation and stress response. An increasing number of studies indicate that SIRTs significantly contribute to the progression of NSCLC by regulating pathophysiological processes such as energy metabolism, autophagy and apoptosis in tumor cells through the deacetylation of histones or non‑histone proteins. The present review elaborates on the roles of different SIRTs and their mechanisms in NSCLC, while also summarizing novel therapeutic agents based on SIRTs. It aims to present new ideas and a theoretical basis for NSCLC treatment.

Enzymatic characterization and dominant sites of foot-and-mouth disease virus 2C protein

Foot-and-mouth disease virus (FMDV) 2C protein is a conserved non-structural protein and crucial for replication of the virus. In this study, FMDV 2C protein was prepared and the enzymatic activities were investigated in detail. The protein could digest ssDNA or ssRNA into a small fragment at about 10 nt, indicating that the protein has nuclease activity. But it did not show digestion to blunt-end dsDNA or dsRNA. The nuclease activity of 2C protein could be inhibited in 2 mM Zn2+ or Ca2+ while enhanced by Mg2+ or Mn2+. FMDV 2C protein exhibited unwinding activity to all the three kinds of dsDNA and dsRNA (5′ protruded, 3′ protruded, and blunt-end). The unwinding velocity to 5′ protruded dsRNA was higher than to the blunt-end dsRNA. 2C protein only showed unwinding activity in high concentration of Mg2+, but no unwinding activity in physiological concentrations of Mg2+ and Ca2+, as well as in cell lysate. The 2C protein could catalyze two structured ssRNA to form double strand, thus it was proved to have RNA chaperone activity. The Mg2+ and ATP in different concentrations did not show promotion to the RNA chaperone activity. Finally, six mutant proteins (K116A, D160A, D170A, N207A, R226A, and F316A) were constructed and the enzymatic activities were analyzed. All the six mutations reduced the ATPase activity, D170A and F361A could inactivate the nuclease activity, while the N207A and F316A could inactivate the helicase activity. Our study provides a comprehensive understanding of the enzymatic activities of FMDV 2C protein.