Caveolin-3 negatively regulates endocytic recycling of cardiac KATP channels.

Sarcolemmal ATP-sensitive potassium (KATP) channels play a vital role in cardioprotection. Cardiac KATP channels are enriched in caveolae and physically interact with the caveolae structural protein caveolin-3 (Cav3). Disrupting caveolae impairs the regulation of KATP channels through several signaling pathways. However, the direct functional effect of Cav3 on KATP channels is still poorly understood. Here, we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and showed that Cav3 greatly reduced KATP channel surface density and current amplitude in a caveolae-independent manner. A screen of Cav3 functional domains revealed that a 25 amino acid region in the membrane attachment domain of Cav3 is the minimal effective segment (MAD1). The peptide corresponding to the MAD1 segment decreased KATP channel current in a concentration-dependent manner with an IC50 of ∼5 µM. The MAD1 segment prevented KATP channel recycling, thus decreasing KATP channel surface density and abolishing the cardioprotective effect of ischemic preconditioning. Our research identified the Cav3 MAD1 segment as a novel negative regulator of KATP channel recycling, providing pharmacological potential in the treatment of diseases with KATP channel trafficking defects.NEW & NOTEWORTHY Cardiac KATP channels physically interact with caveolin-3 in caveolae. In this study, we investigated the functional effect of caveolin-3 on KATP channel activity and identified a novel segment (MAD1) in the C-terminus domain of Caveolin-3 that negatively regulates KATP channel surface density and current amplitude by impairing KATP channel recycling. The peptide corresponding to the MAD1 segment abolished the cardioprotective effect of ischemic preconditioning.

Immune Activation Effects at Different Irradiated Sites and Optimal Timing of Radioimmunotherapy in Patients with Extensive-Stage Small Cell Lung Cancer: a Real-World Analysis.

BACKGROUND: In view of the limited data on radiotherapy (RT) combined with immunotherapy in patients with extensive-stage small cell lung cancer (ES-SCLC), this study aimed to identify the immune activation effect on different sites and the survival outcomes of radioimmunotherapy at different treatment stages. METHODS: Forty-five patients diagnosed with ES-SCLC were included in this retrospective analysis. We collected the overall survival (OS) of the patients,, recorded the blood cell counts before, during, and after RT, and derived blood index ratios such as the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic immune-inflammation index (SII). The datasets were analyzed using the Spearman rank correlation test, Kruskal-Wallis rank sum test and logistic regression. RESULTS: Among the selected blood indices, the delta-NLR/PLR/Sll correlated with different irradiated organs, and the mean ranks of these three indices were the lowest in the brain-irradiated group during immunotherapy. Additionally, adjunct first-line immunotherapy with RT demonstrated a significant improvement compared to second- or third-line therapy and subsequent therapies. CONCLUSION: Our findings suggest that compared to other organs, the strongest immune activation effect occurs with brain RT, and ES-SCLC patients who received radioimmunotherapy (RIT) earlier achieved higher OS rates.

One Stone, Two Birds: A Peptide-Au(I) Infinite Coordination Supermolecule for the Confederate Physical and Biological Radiosensitization in Cancer Radiation Therapy.

Over half of cancer patients are subjected to radiotherapy, but owing to the deficient amount of reactive oxygen radicals (ROS) and DNA double-strand breaks (DSBs), a fair number of them suffer from radiotherapy resistance and the subsequent short-term survival opportunity. To overcome it, many successes have been achieved in radiosensitizer discovery using physical strategy and/or biological strategy, but significant challenges remain regarding developing clinically translational radiosensitizers. Herein, a peptide-Au(I) infinite coordination supermolecule termed PAICS is developed that combined both physical and biological radiosensitization and possessed pharmaceutical characteristics including adequate circulatory stability, controllable drug release, tumor-prioritized accumulation, and the favorable body eliminability. As expected, monovalent gold ion endowed this supermolecule with high X-ray absorption and the subsequent radiosensitization. Furthermore, a peptide targeting CRM1, is assembled into the supermolecule, which successfully activates p53 and apoptosis pathway, thereby further sensitizing radiotherapy. As a result, PAICS showed superior ability for radiotherapy sensitization in vivo and maintained a favorable safety profile. Thus, the PAICS reported here will offer a feasible solution to simultaneously overcome both the pharmaceutical obstacles of physical and biological radiosensitizers and will enable the development of a class of nanomedicines for tumor radiotherapy sensitization.

Hypervalent Iodine Mediated Synthesis of Sulfinamidines from Sulfenamides.

In this study, we present a novel, efficient method for the oxidative amination of sulfenamides using diacetoxyiodobenzene (PhI(OAc)2) and amines under basic conditions. This innovative technique streamlines the synthesis of sulfinamidines under mild, metal-free conditions, achieving outstanding yields of up to 99%. Furthermore, we propose possible pathways that elucidate the observed molecular sequence of events in this reaction. This cutting-edge approach not only advances the synthesis of valuable sulfinamidine compounds but also expands the synthetic toolbox available to chemists, paving the way for future discoveries in organic synthesis and potential applications in medicinal chemistry.

Copper-Catalyzed Aerobic S-Amination of Sulfenamides for the Synthesis of Sulfinamidines.

Herein, we present a copper-catalyzed oxidative amination of sulfenamides for the synthesis of sulfinamidines. By the employment of air as the terminal oxidant, a diverse array of secondary and primary amines can be efficiently transformed into their corresponding products. This method is well-suited for last-stage functionalization, and the underlying mechanism has been investigated. The transformation is characterized by exceptional chemoselectivity, mild conditions, facile operation, and broad substrate compatibility, which have significant implications for the fields of pharmaceuticals and organic synthesis.

Ubiquitin-specific protease 28 deubiquitinates TCF7L2 to govern the action of the Wnt signaling pathway in hepatic carcinoma.

Overexpression of ubiquitin-specific protease 28 (USP28) is found in hepatic carcinoma. It is unclear whether the deubiquitinase plays a role in hepatocarcinogenesis. Deregulation of the Wnt signaling pathway is frequently associated with liver cancer. Transcription factor 7-like 2 (TCF7L2) is an important downstream transcription factor of the Wnt/ß-catenin signaling pathway, but the mechanisms by which TCF7L2 itself is regulated have not yet been revealed. Here, we report that USP28 promotes the activity of the Wnt signaling pathway through maintaining the stability of TCF7L2. We further show that FBXW7 is the E3 ubiquitin ligase for TCF7L2. By regulating the levels of TCF7L2, USP28 modulates the Wnt/ß-catenin signaling in liver cancer and USP28 depletion or inhibition by a small molecule inhibitor leads to a halt of growth in liver cancer cells. These results suggest that USP28 could be a potential therapeutic target for liver cancer.

NADH oxidase of Mycoplasma synoviae is a potential diagnostic antigen, plasminogen/fibronectin binding protein and a putative adhesin.

BACKGROUND: Mycoplasma synoviae (MS) is an important pathogen causing respiratory diseases and arthritis in chickens and turkeys, thus, resulting in serious economic losses to the poultry industry. Membrane-associated proteins are thought to play important roles in cytoadherence and pathogenesis. NADH oxidase (NOX) is an oxidoreductase involved in glycolysis, which is thought to be a multifunctional protein and potential virulence factor in some pathogens. However, little is known regarding the NOX of MS (MSNOX). We previously demonstrated that MSNOX was a metabolic enzyme distributed in not only the cytoplasm but also the MS membrane. This study was aimed at exploring NOX's potential as a diagnostic antigen and its role in MS cytoadherence. RESULTS: Western blots and ELISAs indicated that recombinant MSNOX (rMSNOX) protein reacted with sera positive for various MS isolates, but not MG isolates or other avian pathogens, thus, suggesting that rMSNOX is a potential diagnostic antigen. In addition, rabbit anti-rMSNOX serum showed substantial complement-dependent mycoplasmacidal activity toward various MS isolates and MG Rlow. MSNOX protein was found not only in the cytoplasm but also on the membrane of MS through suspension immunofluorescence and immunogold electron microscopy assays. Indirect immunofluorescence assays indicated that rMSNOX adhered to DF-1 cells, and this adherence was inhibited by rabbit anti-rMSNOX, but not anti-MG serum. Furthermore, indirect immunofluorescence and colony counting assays confirmed that the rabbit anti-rMSNOX serum inhibited the adherence of various MS isolates but not MG Rlow to DF-1 cells. Moreover, plasminogen (Plg)- and fibronectin (Fn)-binding assays demonstrated that rMSNOX bound Plg and Fn in a dose-dependent manner, thereby further confirming that MSNOX may be a putative adhesin. CONCLUSION: MSNOX was identified to be a surface immunogenic protein that has good immunoreactivity and specificity in Western blot and ELISA, and therefore, may be used as a potential diagnostic antigen in the future. In addition, rMSNOX adhered to DF-1 cells, an effect inhibited by rabbit anti-rMSNOX, but not anti-MG serum, and anti-rMSNOX serum inhibited the adherence of various MS isolates, but not MG Rlow, to DF-1 cells, thus indicating that the inhibition of adherence by anti-MSNOX serum was MS specific. Moreover, rMSNOX adhered to extracellular matrix proteins including Plg and Fn, thus suggesting that NOX may play important roles in MS cytoadherence and pathogenesis. Besides, rabbit anti-rMSNOX serum presented complement-dependent mycoplasmacidal activity toward both MS and MG, indicating the MSNOX may be further studied as a potential protective vaccine candidate.

A Novel Immune Gene-Related Prognostic Score Predicts Survival and Immunotherapy Response in Glioma.

Background and Objectives: The clinical prognosis and survival prediction of glioma based on gene signatures derived from heterogeneous tumor cells are unsatisfactory. This study aimed to construct an immune gene-related prognostic score model to predict the prognosis of glioma and identify patients who may benefit from immunotherapy. Methods: 23 immune-related genes (IRGs) associated with glioma prognosis were identified through weighted gene co-expression network analysis (WGCNA) and Univariate Cox regression analysis based on large-scale RNA-seq data. Eight IRGs were retained as candidate predictors and formed an immune gene-related prognostic score (IGRPS) by multifactorial Cox regression analysis. The potential efficacy of immune checkpoint blockade (ICB) therapy of different subgroups was compared by The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm. We further adopted a series of bioinformatic methods to characterize the differences in clinicopathological features and the immune microenvironment between the different risk groups. Finally, a nomogram integrating IGRPS and clinicopathological characteristics was built to accurately predict the prognosis of glioma. Results: Patients in the low-risk group had a better prognosis than those in the high-risk group. Patients in the high-risk group showed higher TIDE scores and poorer responses to ICB therapy, while patients in the low-risk group may benefit more from ICB therapy. The distribution of age and tumor grade between the two subgroups was significantly different. Patients with low IGRPS harbor a high proportion of natural killer cells and are sensitive to ICB treatment. While patients with high IGRPS display relatively poor prognosis, a higher expression level of DNA mismatch repair genes, high infiltrating of immunosuppressive cells, and poor ICB therapeutic outcomes. Conclusions: We demonstrated that the IGRPS model can independently predict the clinical prognosis as well as the ICB therapy responses of glioma patients, thus having important implications on the design of immune-based therapeutic strategies.

Isobavachalcone sensitizes cells to E2-induced paclitaxel resistance by down-regulating CD44 expression in ER+ breast cancer cells.

Oestrogen receptor (ER) is expressed in approximately 60%-70% of human breast cancer. Clinical trials and retrospective analyses have shown that ER-positive (ER+) tumours are more tolerant to chemotherapeutic drug resistance than ER-negative (ER-) tumours. In addition, isobavachalcone (IBC) is known as a kind of phytoestrogen with antitumour effect. However, the underlying mechanism of IBC in ER+ breast cancer needs to be elucidated further. Our in vitro experiments showed that IBC could attenuate 17ß-estradiol (E2 )-induced paclitaxel resistance and that E2 could stimulate CD44 expression in ER+ breast cancer cells but not in ER- cells. Meanwhile, E2 could promote ERα expression to render ER+ breast cancer cells resistant to paclitaxel. Furthermore, we established paclitaxel-resistant breast cancer cell lines and determined the function of ERα in the enhancement of paclitaxel resistance via the regulation of CD44 transcription. IBC down-regulated ERα and CD44 expression and thus inhibited tumour growth in paclitaxel-resistant xenograft models. Overall, our data demonstrated for the first time that IBC could decrease CD44 expression level via the ERα pathway and make ER+ breast cancer cells sensitive to paclitaxel treatment.

Apoptosis and turnover disruption of olfactory sensory neurons in eosinophilic chronic rhinosinusitis.

Olfactory dysfunction (OD) is one of the important and difficult-to-treat symptoms of eosinophilic chronic rhinosinusitis (CRS), which is typically associated with type 2 inflammation where eosinophils (EOSs) function as both effectors and initiators. Eosinophilic infiltration in the olfactory mucosa (OM) is associated with severe OD, mucosal erosion, and more loss of olfactory sensory neurons (OSNs). Active EOS-derived cytokines, chemokines, and eosinophil granule proteins may lead to aggravation of inflammation, tissue damage, and impairment of the survival and regeneration of OSNs. Recent studies show that EOSs can lead to apoptosis of OSNs through axonal and neural body damage, turnover disorder of OSNs through the loss of immature OSNs and globose basal cells (GBCs), changed proliferative activity of horizontal basal cells (HBCs), and dysfunction of OSNs through the breakdown of neuroepithelial integrity and alteration of ion concentration in OSNs and mucin. In this review, we outline the current progress on the role of EOSs on OD in patients with eosinophilic CRS and the mechanism of EOS-associated injury of the OM and OSNs in experimental animal models with sinonasal inflammation. Further investigations on the molecular mechanisms of tissue eosinophilia-induced injury of OSNs are warranted to obtain new therapeutic targets and achieve better restoration of olfactory function.

A novel fatty acid metabolism-related signature identifies MUC4 as a novel therapy target for esophageal squamous cell carcinoma.

Fatty acid metabolism has been identified as an emerging hallmark of cancer, which was closely associated with cancer prognosis. Whether fatty acid metabolism-related genes (FMGs) signature play a more crucial role in biological behavior of esophageal squamous cell carcinoma (ESCC) prognosis remains unknown. Thus, we aimed to identify a reliable FMGs signature for assisting treatment decisions and prognosis evaluation of ESCC. In the present study, we conducted consensus clustering analysis on 259 publicly available ESCC samples. The clinical information was downloaded from The Cancer Genome Atlas (TCGA, 80 ESCC samples) and Gene Expression Omnibus (GEO) database (GSE53625, 179 ESCC samples). A consensus clustering arithmetic was used to determine the FMGs molecular subtypes, and survival outcomes and immune features were evaluated among the different subtypes. Kaplan-Meier analysis and the receiver operating characteristic (ROC) was applied to evaluate the reliability of the risk model in training cohort, validation cohort and all cohorts. A nomogram to predict patients' 1-year, 3-year and 5-year survival rate was also studied. Finally, CCK-8 assay, wound healing assay, and transwell assay were implemented to evaluate the inherent mechanisms of FMGs for tumorigenesis in ESCC. Two subtypes were identified by consensus clustering, of which cluster 2 is preferentially associated with poor prognosis, lower immune cell infiltration. A fatty acid (FA) metabolism-related risk model containing eight genes (FZD10, TACSTD2, MUC4, PDLIM1, PRSS12, BAALC, DNAJA2 and ALOX12B) was established. High-risk group patients displayed worse survival, higher stromal, immune and ESTIMATE scores than in the low-risk group. Moreover, a nomogram revealed good predictive ability of clinical outcomes in ESCC patients. The results of qRT-PCR analysis revealed that the MUC4 and BAALC had high expression level, and FZD10, PDLIM1, TACSTD2, ALOX12B had low expression level in ESCC cells. In vitro, silencing MUC4 remarkably inhibited ESCC cell proliferation, invasion and migration. Our study fills the gap of FMGs signature in predicting the prognosis of ESCC patients. These findings revealed that cluster subtypes and risk model of FMGs had effects on survival prediction, and were expected to be the potential promising targets for ESCC.

Identification of common genetic variants in KCNQ family genes associated with gastric cancer survival in a Chinese population.

KCNQ family genes ( KCNQ1-5), encoding voltage-gated K + (Kv) channels, have been revealed to have potential pathophysiological roles in cancers. However, the associations between genetic variants located in KCNQ family genes and gastric cancer survival remain unclear. A large-scale cohort comprising 1,135 Chinese gastric cancer patients was enrolled to identify genetic variants in KCNQ family genes associated with overall survival (OS). Based on the survival evaluation of all five members, KCNQ1 was selected for subsequent genetic analysis. Cox regression models and stepwise Cox regression models were conducted to evaluate survival-related genetic variants. We found that KCNQ1 rs10832417 was associated with increased OS in gastric cancer patients (adjusted hazard ratio (HR) = 0.84, 95% confidence interval (CI): 0.72-0.98, P = 0.023). Subsequently, a nomogram was generated to support the prognostic capacity and clinical translation of rs10832417 variants. The rs10832417 T allele was predicted to increase the minimum free energy (MFE) of the secondary structure. Furthermore, we observed that gastric cancer patients with downregulation of KCNQ1 had poor survival in multiple public datasets. The present study found that KCNQ1 rs10832417 could serve as an independent prognostic predictor of gastric cancer, yielding novel insight into the progression and survival of gastric cancer.

Delivery of CRISPR/Cas9 system by AAV as vectors for gene therapy.

The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.

Effect of arctigenin on neurological diseases: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Arctium lappa L. is a common specie of Asteraceae. Its main active ingredient, Arctigenin (AG), in mature seeds exerts pharmacological effects on the Central Nervous System (CNS). AIM OF THE STUDY: To review studies on the specific effects of the AG mechanism on various CNS diseases and elucidate signal transduction mechanisms and their pharmacological actions. MATERIALS AND METHODS: This investigation reviewed the essential role of AG in treating neurological disorders. Basic information on Arctium lappa L. was retrieved from the Pharmacopoeia of the People's Republic of China. The related articles from 1981 to 2022 on the network database (including CNKI, PubMed, and Wan Fang and so on) were reviewed using AG and CNS diseases-related terms such as Arctigenin and Epilepsy. RESULTS: It was confirmed that AG has a therapeutic effect on Alzheimer's disease, Glioma, infectious CNS diseases (such as Toxoplasma and Japanese Encephalitis Virus), Parkinson's disease, Epilepsy, etc. In these diseases, related experiments such as a Western blot analysis revealed that AG could alter the content of some key factors (such as the reduction of Aß in Alzheimer's disease). However, in-vivo AG's metabolic process and possible metabolites are still undetermined. CONCLUSION: Based on this review, the existing pharmacological research has indeed made objective progress to elucidate how AG prevents and treats CNS diseases, especially senile degenerative disease such as Alzheimer's diseases. It was revealed that AG could be used as a potential nervous system drug as it has a wide range of effects in theory with markedly high application value, especially in the elder group. However, the existing studies are limited to in-vitro experiments; therefore, little is known about how AG metabolizes and functions in-vivo, limiting its clinical application and requiring further research.

Cocktail hepatocarcinoma therapy by a super-assembled nano-pill targeting XPO1 and ATR synergistically.

Intensive cancer treatment with drug combination is widely exploited in the clinic but suffers from inconsistent pharmacokinetics among different therapeutic agents. To overcome it, the emerging nanomedicine offers an unparalleled opportunity for encapsulating multiple drugs in a nano-carrier. Herein, a two-step super-assembled strategy was performed to unify the pharmacokinetics of a peptide and a small molecular compound. In this proof-of-concept study, the bioinformatics analysis firstly revealed the potential synergies towards hepatoma therapy for the associative inhibition of exportin 1 (XPO1) and ataxia telangiectasia mutated-Rad3-related (ATR), and then a super-assembled nano-pill (gold nano drug carrier loaded AZD6738 and 97-110 amino acids of apoptin (AP) (AA@G)) was constructed through camouflaging AZD6738 (ATR small-molecule inhibitor)-binding human serum albumin onto the AP-Au supramolecular nanoparticle. As expected, both in vitro and in vivo experiment results verified that the AA@G possessed extraordinary biocompatibility and enhanced therapeutic effect through inducing cell cycle arrest, promoting DNA damage and inhibiting DNA repair of hepatoma cell. This work not only provides a co-delivery strategy for intensive liver cancer treatment with the clinical translational potential, but develops a common approach to unify the pharmacokinetics of peptide and small-molecular compounds, thereby extending the scope of drugs for developing the advanced combination therapy.

Ferrostatin-1 attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting ferroptosis.

Background: Hypoxic-ischemic brain damage (HIBD) is a type of brain damage that is caused by perinatal asphyxia and serious damages the central nervous system. At present, there is no effective drug for the treatment of this disease. Besides, the pathogenesis of HIBD remains elusive. While studies have shown that ferroptosis plays an important role in HIBD, its role and mechanism in HIBD are yet to be fully understood. Methods: The HIBD model of neonatal rats was established using the Rice-Vannucci method. A complete medium of PC12 cells was adjusted to a low-sugar medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 12 h. Laser Doppler blood flow imaging was used to detect the blood flow intensity after modeling. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining and transmission electron microscopy were used to observe brain injury and mitochondrial damage. Immunofluorescence was applied to monitor the expression of GFAP. Real-time quantitative polymerase chain reaction, western blot, and immunofluorescence were utilized to detect the expression of messenger RNA and protein. The level of reactive oxygen species (ROS) in cells was detected using the ROS detection kit. Results: The results showed that ferrostatin-1 (Fer-1) significantly alleviated the brain injury caused by hypoxia and ischemia. Fer-1 significantly increased the expression of SLC3A2, SLC7A11, ACSL3, GSS, and GPX4 (P<0.05) and dramatically decreased the expressions of GFAP, ACSL4, TFRC, FHC, FLC, 4-HNE, HIF-1α, and ROS (P<0.05). Conclusions: Fer-1 inhibits ferroptosis and alleviates HIBD by potentially targeting the GPX4/ACSL3/ACSL4 axis; however, its specific mechanism warrants further exploration.

Rationale, Design and Participants Baseline Characteristics of a Crossover Randomized Controlled Trial of the Effect of Replacing SSBs with NSBs versus Water on Glucose Tolerance, Gut Microbiome and Cardiometabolic Risk in Overweight or Obese Adult SSB Consumer: Strategies to Oppose SUGARS with Non-Nutritive Sweeteners or Water (STOP Sugars NOW) Trial and Ectopic Fat Sub-Study.

BACKGROUND: Health authorities are near universal in their recommendation to replace sugar-sweetened beverages (SSBs) with water. Non-nutritive sweetened beverages (NSBs) are not as widely recommended as a replacement strategy due to a lack of established benefits and concerns they may induce glucose intolerance through changes in the gut microbiome. The STOP Sugars NOW trial aims to assess the effect of the substitution of NSBs (the "intended substitution") versus water (the "standard of care substitution") for SSBs on glucose tolerance and microbiota diversity. DESIGN AND METHODS: The STOP Sugars NOW trial (NCT03543644) is a pragmatic, "head-to-head", open-label, crossover, randomized controlled trial conducted in an outpatient setting. Participants were overweight or obese adults with a high waist circumference who regularly consumed ≥1 SSBs daily. Each participant completed three 4-week treatment phases (usual SSBs, matched NSBs, or water) in random order, which were separated by ≥4-week washout. Blocked randomization was performed centrally by computer with allocation concealment. Outcome assessment was blinded; however, blinding of participants and trial personnel was not possible. The two primary outcomes are oral glucose tolerance (incremental area under the curve) and gut microbiota beta-diversity (weighted UniFrac distance). Secondary outcomes include related markers of adiposity and glucose and insulin regulation. Adherence was assessed by objective biomarkers of added sugars and non-nutritive sweeteners and self-report intake. A subset of participants was included in an Ectopic Fat sub-study in which the primary outcome is intrahepatocellular lipid (IHCL) by 1H-MRS. Analyses will be according to the intention to treat principle. BASELINE RESULTS: Recruitment began on 1 June 2018, and the last participant completed the trial on 15 October 2020. We screened 1086 participants, of whom 80 were enrolled and randomized in the main trial and 32 of these were enrolled and randomized in the Ectopic Fat sub-study. The participants were predominantly middle-aged (mean age 41.8 ± SD 13.0 y) and had obesity (BMI of 33.7 ± 6.8 kg/m2) with a near equal ratio of female: male (51%:49%). The average baseline SSB intake was 1.9 servings/day. SSBs were replaced with matched NSB brands, sweetened with either a blend of aspartame and acesulfame-potassium (95%) or sucralose (5%). CONCLUSIONS: Baseline characteristics for both the main and Ectopic Fat sub-study meet our inclusion criteria and represent a group with overweight or obesity, with characteristics putting them at risk for type 2 diabetes. Findings will be published in peer-reviewed open-access medical journals and provide high-level evidence to inform clinical practice guidelines and public health policy for the use NSBs in sugars reduction strategies. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT03543644.

Research on improvement of DPoS consensus mechanism in collaborative governance of network public opinion.

With the increasingly complex social situation, the problems of traditional online public opinion governance are increasingly serious. Especially the problem of transmission efficiency, public opinion data management and user information security of Internet users is urgently needed. Here, we design a functional infrastructure framework of the network public opinion collaborative governance model based on the blockchain with strong practicality and comprehensiveness. In order to reach the consensus mechanism requirements under the framework, the algorithm is improved on the basis of the defects of the traditional DPoS consensus algorithm. Considering time dynamic factors in the process of reaching consensus, the paper proposes a reputation-based voting model. Furthermore, the paper purposes a rewards and punishments incentive mechanism, and also designs a new method of counting votes. From the simulation results, it was found that after the improvement of the algorithm, the enthusiasm of node participation was significantly increased, the proportion of error nodes was significantly reduced, and the operating efficiency was significantly improved. It shows that the improved consensus algorithm we propose applies to public opinion governance can not only improve the security of the system with the reduce of false public opinion spreading, but also improve the efficiency of information processing, so it can be well applied to information sharing and public opinion governance scenarios.

Immune-Associated Gene Signatures Serve as a Promising Biomarker of Immunotherapeutic Prognosis for Renal Clear Cell Carcinoma.

As the most common type of renal cell carcinoma (RCC), the renal clear cell carcinoma (ccRCC) is highly malignant and insensitive to chemotherapy or radiotherapy. Although systemic immunotherapies have been successfully applied to ccRCC in recent years, screening for patients who can benefit most from these therapies is still essential and challenging due to immunological heterogeneity of ccRCC patients. To this end, we implemented a series of deep investigation on the expression and clinic data of ccRCC from The Cancer Genome Atlas (TCGA) International Consortium for Cancer Genomics (ICGC). We identified a total of 946 immune-related genes that were differentially expressed. Among them, five independent genes, including SHC1, WNT5A, NRP1, TGFA, and IL4R, were significantly associated with survival and used to construct the immune-related prognostic differential gene signature (IRPDGs). Then the ccRCC patients were categorized into high-risk and low-risk subgroups based on the median risk score of the IRPDGs. IRPDGs subgroups displays distinct genomic and immunological characteristics. Known immunotherapy-related genes show different mutation burden, wherein the mutation rate of VHL was higher than 40% in the two IRPDGs subgroups, and SETD2 and BAP1 mutations differed most between two groups with higher frequency in the high-risk subgroup. Moreover, IRPDGs subgroups had different abundance in tumor-infiltrating immune cells (TIICs) with distinct immunotherapy efficacy. Plasma cells, regulatory cells (Tregs), follicular helper T cells (Tfh), and M0 macrophages were enriched in the high-risk group with a higher tumor immune dysfunction and rejection (TIDE) score. In contrast, the low-risk group had abundant M1 macrophages, mast cell resting and dendritic cell resting infiltrates with lower TIDE score and benefited more from immune checkpoint inhibitors (ICI) treatment. Compared with other biomarkers, such as TIDE and tumor inflammatory signatures (TIS), IRPDGs demonstrated to be a better biomarker for assessing the prognosis of ccRCC and the efficacy of ICI treatment with the promise in screening precise patients for specific immunotherapies.

A Novel Necroptosis-Related lncRNA Signature Predicts the Prognosis of Lung Adenocarcinoma.

Background: Necroptosis is closely related to the tumorigenesis and development of cancer. An increasing number of studies have demonstrated that targeting necroptosis could be a novel treatment strategy for cancer. However, the predictive potential of necroptosis-related long noncoding RNAs (lncRNAs) in lung adenocarcinoma (LUAD) still needs to be clarified. This study aimed to construct a prognostic signature based on necroptosis-related lncRNAs to predict the prognosis of LUAD. Methods: We downloaded RNA sequencing data from The Cancer Genome Atlas database. Co-expression network analysis, univariate Cox regression, and least absolute shrinkage and selection operator were adopted to identify necroptosis-related prognostic lncRNAs. We constructed the predictive signature by multivariate Cox regression. Kaplan-Meier analysis, time-dependent receiver operating characteristics, nomogram, and calibration curves were used to validate and evaluate the signature. Subsequently, we used gene set enrichment analysis (GSEA) and single-sample gene set enrichment analysis (ssGSEA) to explore the relationship between the predictive signature and tumor immune microenvironment of risk groups. Finally, the correlation between the predictive signature and immune checkpoint expression of LUAD patients was also analyzed. Results: We constructed a signature composed of 7 necroptosis-related lncRNAs (AC026355.2, AC099850.3, AF131215.5, UST-AS2, ARHGAP26-AS1, FAM83A-AS1, and AC010999.2). The signature could serve as an independent predictor for LUAD patients. Compared with clinicopathological variables, the necroptosis-related lncRNA signature has a higher diagnostic efficiency, with the area under the receiver operating characteristic curve being 0.723. Meanwhile, when patients were stratified according to different clinicopathological variables, the overall survival of patients in the high-risk group was shorter than that of those in the low-risk group. GSEA showed that tumor- and immune-related pathways were mainly enriched in the low-risk group. ssGSEA further confirmed that the predictive signature was significantly related to the immune status of LUAD patients. The immune checkpoint analysis displayed that low-risk patients had a higher immune checkpoint expression, such as CTLA-4, HAVCR2, PD-1, and TIGIT. This suggested that immunological function is more active in the low-risk group LUAD patients who might benefit from checkpoint blockade immunotherapies. Conclusion: The predictive signature can independently predict the prognosis of LUAD, helps elucidate the mechanism of necroptosis-related lncRNAs in LUAD, and provides immunotherapy guidance for patients with LUAD.