The ubiquitin ligase STUB1 suppresses tumorigenesis of renal cell carcinomas through regulating YTHDF1 stability.

STIP1 homology and U-box protein 1 (STUB1), a crucial member of the RING family E3 ubiquitin ligase, serves dual roles as an oncogene and a tumor suppressor in various human cancers. However, the role and mechanism of STUB1 in clear cell renal cell carcinoma (ccRCC) remain poorly defined. Here, we identified YTHDF1 as a novel STUB1 interaction partner using affinity purification mass spectrometry (AP-MS). Furthermore, we revealed that STUB1 promotes the ubiquitination and degradation of YTHDF1. Consequently, STUB1 depletion leads to YTHDF1 up-regulation in renal cancer cells. Functionally, STUB1 depletion promoted migration and invasion of ccRCC cells in a YTHDF1-dependent manner. Additionally, depletion of STUB1 also increased the tumorigenic potential of ccRCC in a xenograft model. Importantly, STUB1 expression is down-regulated in ccRCC tissues, and its low expression level correlates with advanced tumor stage and poor overall survival in ccRCC patients. Taken together, these findings reveal that STUB1 inhibits the tumorigenicity of ccRCC by regulating YTHDF1 stability.

A developed and validated centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method for rapid quantification of unbound lenvatinib in human plasma.

In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (Vu). The results indicated that the centrifugal force and centrifugal time have an influence on Vu that is significantly positive (P < 0.05). This developed method with good linearity (r2 = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %-106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice.

Epigenetic regulation of FOXI2 promotes clear cell renal cell carcinoma progression.

In recent decades, substantial advancements in epigenetics have unveiled a profound understanding of its mechanisms in tumorigenesis and have offered promising strategies for epigenetic therapy in cancer patients. In our study, through bioinformatics analysis, we discovered a significant downregulation and hypermethylation of FOXI2 in clear cell renal cell carcinoma (ccRCC), while the expression in chromophobe cell carcinoma (chRCC) exhibited the opposite trend. Moreover, we established a strong correlation between FOXI2 expression levels and the prognosis of ccRCC. Gene enrichment analysis and cell function experiments unequivocally demonstrate that FOXI2 possesses the capability to induce cell cycle arrest and inhibit cell proliferation. Our research findings demonstrate that the expression of FOXI2 in ccRCC is under the regulation of promoter hypermethylation. Furthermore, in vitro experiments have conclusively shown that the overexpression of FOXI2 induces cell cycle arrest and inhibits cell proliferation.

Effects of different emergent macrophytes on methane flux and rhizosphere microbial communities in wetlands.

Emergent macrophytes are of great importance for the structure and functioning of wetland ecosystems and play a significant role in environmental improvement, element cycling, and greenhouse gas (GHG) emissions. However, our understanding of how GHG fluxes differ among macrophyte species and its links with the microbial communities remain limited. In this study, we investigated the rhizosphere microbial communities (including total bacteria, methanotrophs, and methanogens) and the GHG fluxes associated with four emergent macrophytes-Phragmites australis, Thalia dealbata, Pontederia cordata, and Zizania latifolia-collected from Xuanwu Lake wetland, China. We observed the highest CH4 flux (FCH4) (9.35 ± 2.52 mg·m-2·h-1) from Z. latifolia zone, followed by P. australis, P. cordata, and T. dealbata zones (5.38 ± 1.63, 2.38 ± 2.91, and 2.02 ± 0.69 mg·m-2·h-1, respectively). Methanogenesis was methylotrophic at all sites, as the 13C-CH4 values were higher than -64 ‰ and the fractionation coefficients were lower than 1.055. We found a positive linear relationship between FCH4 and the methanogen community, in particular the relative abundances of Methanobacterium and Methanosarcina, indicating that the variations in FCH4 among the studied macrophyte-dominated zones might be attributed to the differences in rhizosphere microbial communities. The methane emissions in various macrophyte zones might be due to the higher capacity of methanogenesis compared to methane oxidation which was inhibited by nutrient-rich sediments. Our findings provide insights for selecting specific emergent macrophytes characterized by low FCH4 in wetland ecological restoration.

Synergistic antibacterial and antifouling wound dressings: Integration of photothermal-activated no release and zwitterionic surface modification.

Addressing the pervasive issue of bacteria and biofilm infections is crucial in the development of advanced antifouling wound dressings. In this study, a novel wound healing treatment using sulfobetaine (SBMA) decorated electrospun fibrous membrane based on polycaprolactone (PCL)/nitric oxide (NO) donors was developed. The fabrication involved a dual strategy, first integrating NO donors into mesoporous polydopamine (MPDA) and complexed with PCL/PEI to electrospin nanofibers. The fibrous membrane exhibited a potent antibacterial response upon irradiation at 808 nm, owing to a combination of NO and photothermal effect that effectively targets bacteria and disrupts biofilms. Surface functionalization of the membrane with PEI allowed for the attachment of SBMA via Michael addition, fabricating a zwitterionic surface, which significantly hinders protein adsorption and reduces biofilm formation on the wound dressing. In vitro and in vivo assessments confirmed the rapid bactericidal capabilities and its efficacy in biofilm eradication. Combining photothermal activity, targeted NO release and antifouling surface, this multifaceted wound dressing addresses key challenges in bacterial infection management and biofilm eradication, promoting efficient wound healing.

Inhibitory effects of black phosphorus nanosheets on tumor cell proliferation through downregulation of ADIPOQ and downstream signaling pathways.

Exposure to environmental pollutants, including nanomaterials, has a significant impact on tumor progression. The increased demand for black phosphorus nanosheets (BPNSs), driven by their exceptional properties, raises concerns about potential environmental contamination. Assessing their toxicity on tumor growth is essential. Herein, we employed a range of biological techniques, including cytotoxicity measurement, bioinformatics tools, proteomics, target gene overexpression, Western blot analysis, and apoptosis detection, to investigate the toxicity of BPNSs across A549, HepG-2, MCF-7, and Caco-2 cell lines. Our results demonstrated that BPNSs downregulated the expression of ADIPOQ and its associated downstream pathways, such as AMP-activated protein kinase (AMPK), nuclear factor erythroid 2-related factor 2 (Nrf2), and other unidentified pathways. These downregulated pathways ultimately led to mitochondria-dependent apoptosis. Notably, the specific downstream pathways involved varied depending on the type of tumors. These insightful findings not only confirm the consistent inhibitory effects of BPNSs across different tumor cells, but also elucidate the cytotoxicity mechanisms of BPNSs in different tumors, providing valuable information for their safe application and health risk assessment.

The enigmatic interplay of immune cells and abnormal spermatozoa through Mendelian randomization.

PURPOSE: Abnormal spermatozoa significantly impact reproductive health, affecting fertility rates, potentially prolonging conception time, and increasing the risk of miscarriages. This study employs Mendelian randomization to explore their potential link with immune cells, aiming to reveal their potential causal association and wider implications for reproductive health. METHODS: We conducted forward and reverse Mendelian randomization analyses to explore the potential causal connection between 731 immune cell signatures and abnormal spermatozoa. Using publicly available genetic data, we investigated various immune signatures such as median fluorescence intensities (MFI), relative cell (RC), absolute cell (AC), and morphological parameters (MP). Robustness was ensured through comprehensive sensitivity analyses assessing consistency, heterogeneity, and potential horizontal pleiotropy. The MR study produced a statistically significant p-value of .0000684, Bonferroni-corrected for the 731 exposures. RESULTS: The Mendelian randomization analysis revealed strong indications of a reciprocal relationship between immune cell pathways and sperm integrity. When examining immune cell exposure, a potential causal link with abnormal sperm was observed in 35 different types of immune cells. Conversely, the reverse Mendelian randomization results indicated that abnormal sperm might causally affect 39 types of immune cells. These outcomes suggest a potential mutual influence between alterations in immune cell functionality and the quality of spermatozoa. CONCLUSION: This study highlights the close link between immune responses and sperm development, suggesting implications for reproductive health and immune therapies. Further research may offer crucial insights into male fertility and immune disorders.

Accurate prediction of myopic progression and high myopia by machine learning.

Background: Myopia is a leading cause of visual impairment in Asia and worldwide. However, accurately predicting the progression of myopia and the high risk of myopia remains a challenge. This study aims to develop a predictive model for the development of myopia. Methods: We first retrospectively gathered 612 530 medical records from five independent cohorts, encompassing 227 543 patients ranging from infants to young adults. Subsequently, we developed a multivariate linear regression algorithm model to predict the progression of myopia and the risk of high myopia. Result: The model to predict the progression of myopia achieved an R2 value of 0.964 vs a mean absolute error (MAE) of 0.119D [95% confidence interval (CI): 0.119, 1.146] in the internal validation set. It demonstrated strong generalizability, maintaining consistent performance across external validation sets: R2 = 0.950 vs MAE = 0.119D (95% CI: 0.119, 1.136) in validation study 1, R2 = 0.950 vs MAE = 0.121D (95% CI: 0.121, 1.144) in validation study 2, and R2 = 0.806 vs MAE = -0.066D (95% CI: -0.066, 0.569) in the Shanghai Children Myopia Study. In the Beijing Children Eye Study, the model achieved an R2 of 0.749 vs a MAE of 0.178D (95% CI: 0.178, 1.557). The model to predict the risk of high myopia achieved an area under the curve (AUC) of 0.99 in the internal validation set and consistently high area under the curve values of 0.99, 0.99, 0.96 and 0.99 in the respective external validation sets. Conclusion: Our study demonstrates accurate prediction of myopia progression and risk of high myopia providing valuable insights for tailoring strategies to personalize and optimize the clinical management of myopia in children.

Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Unraveling the role of M1 macrophage and CXCL9 in predicting immune checkpoint inhibitor efficacy through multicohort analysis and single-cell RNA sequencing.

The exact function of M1 macrophages and CXCL9 in forecasting the effectiveness of immune checkpoint inhibitors (ICIs) is still not thoroughly investigated. We investigated the potential of M1 macrophage and C-X-C Motif Chemokine Ligand 9 (CXCL9) as predictive markers for ICI efficacy, employing a comprehensive approach integrating multicohort analysis and single-cell RNA sequencing. A significant correlation between high M1 macrophage and improved overall survival (OS) and objective response rate (ORR) was found. M1 macrophage expression was most pronounced in the immune-inflamed phenotype, aligning with increased expression of immune checkpoints. Furthermore, CXCL9 was identified as a key marker gene that positively correlated with M1 macrophage and response to ICIs, while also exhibiting associations with immune-related pathways and immune cell infiltration. Additionally, through exploring RNA epigenetic modifications, we identified Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3G (APOBEC3G) as linked to ICI response, with high expression correlating with improved OS and immune-related pathways. Moreover, a novel model based on M1 macrophage, CXCL9, and APOBEC3G-related genes was developed using multi-level attention graph neural network, which showed promising predictive ability for ORR. This study illuminates the pivotal contributions of M1 macrophages and CXCL9 in shaping an immune-active microenvironment, correlating with enhanced ICI efficacy. The combination of M1 macrophage, CXCL9, and APOBEC3G provides a novel model for predicting clinical outcomes of ICI therapy, facilitating personalized immunotherapy.

Crosstalk between ubiquitin ligases and ncRNAs drives cardiovascular disease progression.

Cardiovascular diseases (CVDs) are multifactorial chronic diseases and have the highest rates of morbidity and mortality worldwide. The ubiquitin-proteasome system (UPS) plays a crucial role in posttranslational modification and quality control of proteins, maintaining intracellular homeostasis via degradation of misfolded, short-lived, or nonfunctional regulatory proteins. Noncoding RNAs (ncRNAs, such as microRNAs, long noncoding RNAs, circular RNAs and small interfering RNAs) serve as epigenetic factors and directly or indirectly participate in various physiological and pathological processes. NcRNAs that regulate ubiquitination or are regulated by the UPS are involved in the execution of target protein stability. The cross-linked relationship between the UPS, ncRNAs and CVDs has drawn researchers' attention. Herein, we provide an update on recent developments and perspectives on how the crosstalk of the UPS and ncRNAs affects the pathological mechanisms of CVDs, particularly myocardial ischemia/reperfusion injury, myocardial infarction, cardiomyopathy, heart failure, atherosclerosis, hypertension, and ischemic stroke. In addition, we further envision that RNA interference or ncRNA mimics or inhibitors targeting the UPS can potentially be used as therapeutic tools and strategies.

Glucuronidation dynamics of curcumin and tetrahydrocurcumin for differential structures and chemical reactivities in human liver microsome and uridine diphosphate glucuronosyltransferase 2B7.

THC is the main metabolite of curcumin with better bioactivity. This study aimed to explore the factors that cause differences in the bioactivity of curcumin and THC. We analyzed the metabolic activities of curcumin and THC and the factors responsible for the differences in their activities by glucuronidation activity assay, LC-MS, HPLC, homologous sequence comparisons, and molecular docking. Curcumin has higher metabolic activity than THC in HLM and UGT2B7, while the keto-enol isomers of curcumin and THC were distinctly different under different pH, and their structural transformations were hypothesized. Furthermore, UGT1A and UGT2B are differential sequences of curcumin and THC in UGTs. The binding sites and patterns of curcumin and THC in UGT2B7 are markedly different. In summary, the difference in keto-enolic interconversion isomerism between curcumin and THC is the main factor causing the difference in their activities, which provides a scientific basis for the development of curcumin.

Understanding Rejection Mechanisms of Trace Organic Contaminants by Polyamide Membranes via Data-Knowledge Codriven Machine Learning.

Data-driven machine learning (ML) provides a promising approach to understanding and predicting the rejection of trace organic contaminants (TrOCs) by polyamide (PA). However, various confounding variables, coupled with data scarcity, restrict the direct application of data-driven ML. In this study, we developed a data-knowledge codriven ML model via domain-knowledge embedding and explored its application in comprehending TrOC rejection by PA membranes. Domain-knowledge embedding enhanced both the predictive performance and the interpretability of the ML model. The contribution of key mechanisms, including size exclusion, charge effect, hydrophobic interaction, etc., that dominate the rejections of the three TrOC categories (neutral hydrophilic, neutral hydrophobic, and charged TrOCs) was quantified. Log D and molecular charge emerge as key factors contributing to the discernible variations in the rejection among the three TrOC categories. Furthermore, we quantitatively compared the TrOC rejection mechanisms between nanofiltration (NF) and reverse osmosis (RO) PA membranes. The charge effect and hydrophobic interactions possessed higher weights for NF to reject TrOCs, while the size exclusion in RO played a more important role. This study demonstrated the effectiveness of the data-knowledge codriven ML method in understanding TrOC rejection by PA membranes, providing a methodology to formulate a strategy for targeted TrOC removal.

Light Scattering Method for Aerosol Sizing Based on Machine Learning.

Optical scattering has been widely used for aerosol sizing due to its noninvasive and real-time measurement. However, it is crucial to retrieve the particle size distribution (PSD) of aerosols without prior knowledge of the refractive index. Now, it has been a great challenge to measure the refractive index in situ. In this study, a novel PSD sensing method utilizing the light scattering angular spectrum (LSAS) and machine learning techniques is proposed to address this challenge. The complex nonlinear relationship between LSAS and PSD can be constructed while accounting for the refractive index of aerosols. A miniaturized prototype sensor is designed and tested on different sizes of aerosol samples. The experiment results showed that the maximum Kullback-Leibler divergence (DKL) of PSD is 0.07, which indicates that the sensing method can provide the ability for highly accurate aerosol PSD measurement without requiring prior knowledge of the refractive index. The compacted prototype sensor shows great potential for aerosol analysis in conventional field measurements outside the laboratory.

The global distribution of special needs dentistry across dental school curricula.

INTRODUCTION: Special needs dentistry (SND) is an emerging dental specialty, with ongoing developments in education and clinical practice focused towards the tailored management of individuals with special needs (SN). Patients with SN have a higher prevalence of oral diseases and unmet dental needs compared to the general population. Although inadequate training and experience in managing patients with SN has been highlighted as a significant barrier to accessing care, there is limited data about the extent of SND teaching at the entry-to-practice or higher levels. METHODS: This work is the first to map SND curricula globally, across 180 countries and 1265 dental schools. RESULTS: Although 74.62% of dental schools were found in developing economies, the distribution of programs that reported SND in their courses was highly skewed towards developed countries. In terms of advanced degrees, beyond basic entry-to-practice training, the USA delivered 60% of the SND programs, followed by Canada (15.56%), UK (13.33%), and Australia (8.89%). The term SND appeared in 33.95% of entry-to-practice level program curricula and was less commonly used in transitioning economies. Only 112 SND-specialized practitioners enter the workforce globally each year from developed economies, and all but three advanced degrees are found in G7 countries. CONCLUSION: By exploring the impact of economic status on its distribution, this paper highlighted the lack of SND representation in dental curricula, especially amongst programs in transitioning or developing economies. Education of both general dentists and specialists is critical as a collaborative effort is needed to manage the growing population of patients with SN.

Microbiome: Role in Inflammatory Skin Diseases.

As the body's largest organ, the skin harbors a highly diverse microbiota, playing a crucial role in resisting foreign pathogens, nurturing the immune system, and metabolizing natural products. The dysregulation of human skin microbiota is implicated in immune dysregulation and inflammatory responses. This review delineates the microbial alterations and immune dysregulation features in common Inflammatory Skin Diseases (ISDs) such as psoriasis, rosacea, atopic dermatitis(AD), seborrheic dermatitis(SD), diaper dermatitis(DD), and Malassezia folliculitis(MF).The skin microbiota, a complex and evolving community, undergoes changes in composition and function that can compromise the skin microbial barrier. These alterations induce water loss and abnormal lipid metabolism, contributing to the onset of ISDs. Additionally, microorganisms release toxins, like Staphylococcus aureus secreted α toxins and proteases, which may dissolve the stratum corneum, impairing skin barrier function and allowing entry into the bloodstream. Microbes entering the bloodstream activate molecular signals, leading to immune disorders and subsequent skin inflammatory responses. For instance, Malassezia stimulates dendritic cells(DCs) to release IL-12 and IL-23, differentiating into a Th17 cell population and producing proinflammatory mediators such as IL-17, IL-22, TNF-α, and IFN-α.This review offers new insights into the role of the human skin microbiota in ISDs, paving the way for future skin microbiome-specific targeted therapies.

Methane emissions and microbial communities under differing flooding conditions and seasons in littoral wetlands of urban lake.

Wetlands are the largest natural sources of methane (CH4) emissions worldwide. Littoral wetlands of urban lakes represent an ecotone between aquatic and terrestrial ecosystems and are strongly influenced by water levels, environmental conditions, and anthropogenic activities. Despite these littoral zones being potential "hotspots" of CH4 emissions, the status of CH4 emissions therein and the role of physicochemical properties and microbial communities regulating these emissions remain unclear. This study compared the CH4 fluxes, physicochemical properties, and CH4-cycling microbial communities (methanogens and methanotrophs) of three zones (a non-flooded supralittoral zone, a semi-flooded eulittoral zone, and a flooded infralittoral zone) in the littoral wetlands of Lake Pipa, Jiangsu Province, China, for two seasons (summer and winter). The eulittoral zone was a CH4 source (median: 11.49 and 0.02 mg m-2 h-1 in summer and winter, respectively), whereas the supralittoral zone acted as a CH4 sink (median: -0.78 and -0.09 mg m-2 h-1 in summer and winter, respectively). The infralittoral zone shifted from CH4 sink to source between the summer (median: -10.65 mg m-2 h-1) and winter (median: 0.11 mg m-2 h-1). The analysis of the functional genes of methanogenesis (mcrA) and methanotrophy (pmoA) and path analysis showed that CH4 fluxes were strongly regulated by biotic factors (abundance of the mcrA gene and alpha diversity of CH4-cycling microbial communities) and abiotic factors (ammonia nitrogen, moisture, and soil organic carbon). In particular, biotic factors had a major influence on the variation in the CH4 flux, whereas abiotic factors had a minor influence. Our findings provide novel insights into the spatial and seasonal variations in CH4-cycling microbial communities and identify the key factors influencing CH4 fluxes in littoral wetlands. These results are important for managing nutrient inputs and regulating the hydrological regimes of urban lakes.

Gut Microbiota Disorders in Obesity-Associated Benign Prostatic Hyperplasia in Rats.

Benign prostatic hyperplasia (BPH), commonly seen in older men, can cause symptoms of discomfort, and may even need surgical intervention. Studies have shown the potential link between gut microbes and BPH, but the molecular association is not fully understood. METHODS: Four-week-old male Sprague-Dawley rats (n = 16) were randomly allocated to normal control diet (ND, 10% fat) and high-fat diet-induced BPH (HFD, 45% fat) groups. Metagenomic analysis was used to examine the abundance and discrepancies in gut microbiota within the two groups after 24 weeks of feeding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted to assess the biological functions of the differentially expressed genes. RESULTS: Rats with HFD-induced obesity exhibited morphological abnormalities in their prostate tissues. Metagenomic analysis of the gut revealed that Firmicutes were the dominant phyla in the HFD group, whereas the ND group had a higher abundance of Spirochaetes. At the genus level, Ruminococcus spp exhibited greater abundance in the HFD group, whereas Treponema spp were more abundant in the ND group. KEGG analysis demonstrated that the differentially expressed genes were mainly enriched in the NOD-like receptor (NLR) signaling, PI3K-Akt signaling, estrogen-signaling, signalings associated with GABAergic synapses, pantothenate and CoA biosynthesis. CONCLUSION: The findings of our study indicated that there was a notable variation in the microbiota abundance within the intestinal tract of obese rats suffering from prostate hyperplasia. It is plausible that these differentially abundant bacteria played a role in the development of pathological alterations in the prostate through the facilitation of inflammatory responses; however, additional research is required to validate the findings.

Role of heat shock protein 70 in silibinin-induced apoptosis in bladder cancer.

Hsp70 (heat shock protein 70) plays critical roles in cancer cell proliferation and apoptosis. Recently, accumulating evidences have demonstrated the cancer promoting effects of Hsp70 in bladder cancer. The development of novel therapeutic approaches targeting Hsp70 thus received great attention from researchers. In this study, we demonstrated that silibinin, a natural polyphenolic flavonoid isolated from the milk thistle, targeted Hsp70 by inhibiting its transcription in bladder cancer cells. We also demonstrated that knockdown of endogenous Hsp70 enhanced silibinin-induced apoptosis, while overexpression of exogenous Hsp70 could partially reverse the effects of silibinin-induced cell apoptosis. Furthermore, we found that silibinin could activate HSF1/Hsp70-regulated mitochondrial apoptotic pathway. Mechanically, silibinin inhibited the interaction between Apaf-1 and Hsp70, thus increasing the recruitment of pro caspase-9. Results from in vivo study demonstrated that silibinin suppressed the growth of bladder cancer xenografts, which was accompanied with the activation of caspase-3 and downregulation of HSF1 and Hsp70. Taken together, our data indicates that silibinin induces mitochondrial apoptosis via inhibiting HSF1/Hsp70 pathway and also suggests the therapeutic potential of silibinin in the treatment of bladder cancer.

Establishment of an Indirect ELISA Method for the Detection of the Bovine Rotavirus VP6 Protein.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 µg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.