Nitrocellulose membranes in situ grown with Prussian blue nanoparticles as stable nanozyme pads for colorimetric detection of dopamine.

Prussian blue (PB) is a typical peroxidase mimic with simple preparation, low cost and high eco-friendliness, but it still has drawbacks of poor stability (e.g., decomposition in aqueous dispersions) and intrinsic optical interference (e.g., high extinction coefficient over a wide wavelength range) in colorimetric assays. Herein, we used nitrocellulose (NC) membranes as synthesis hosts of PB nanoparticles (NPs) to develop a new type of three-dimensional (3D) porous nanozyme pad. By means of an in situ synthesis route, PB NPs were uniformly grown on the surfaces of the fiber scaffolds with desirable stability, which also avoided signal interference from PB NPs owing to the easy handling of the pads in a quantitative solid state. The pads showed significant peroxidase-mimicking activity toward the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with the output of colorimetric signals. Based on the reduction of oxidized TMB (oxTMB) by dopamine (DA), the pads were exploited for simple and quantitative colorimetric detection of DA with a limit of detection (LOD) of 0.59 µM and a satisfactory accuracy for analysis of real human urine samples. This is the first attempt at exploiting NC membranes as the synthesis hosts to develop nanozyme pads, which solves the above drawbacks of traditional PB-based peroxidase mimics and has promise for various colorimetric bioanalyses, given the structural benefits of NC membranes and their broad applications in biosensors.

Role of TFEB in regulation of the podocyte actin cytoskeleton.

Podocyte injury is linked to the pathogenesis and progression of renal disease. The Transcription Factor EB (TFEB), a master regulator of the autophagy and lysosomal pathways, has been found to exert cell- and tissue-specific biological function. To explore TFEB function and underlying mechanisms in podocytes, a total of 4645 differentially expressed genes (DEGs) were detected in TFEB-knockdown mouse podocytes by transcriptome sequencing. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Ingenuity Pathway Analysis showed that, apart from the enrichment in autophagy and lysosomal pathways, DEGs were enriched in cytoskeleton structure (Actin Cytoskeleton, Focal Adhesion, and Adherens Junction), as well as cytoskeleton regulatory molecular signaling (Hippo and Rho GTPase Signaling). In vitro, TFEB knockdown resulted in podocyte cytoskeletal rearrangement, which was disorganized with cortical distribution of actin filaments. Further, TFEB knockdown decreased mRNA and protein levels of Synaptopodin and led to the rearrangement of Synaptopodin. Inhibition of TFEB decreased mRNA levels for proteins involved in actin cytoskeleton dynamics. Moreover, apoptosis was increased by TFEB knockdown in podocyte. In summary, this study initiated a comprehensive analysis of the role of TFEB in podocyte function and the potential underlying mechanisms, and identified a novel role for TFEB in regulation of the podocyte actin cytoskeleton.

Growth associated protein 43 deficiency promotes podocyte injury by activating the calmodulin/calcineurin pathway under hyperglycemia.

Podocyte injury is a crucial factor in the pathogenesis of diabetic kidney disease (DKD), and finding potential therapeutic interventions that can mitigate podocyte injury holds significant clinical relevance. This study was to elucidate the role of growth associated protein-43(Gap43) in podocyte injury of high glucose (HG). We confirmed the expression of Gap43 in human glomerulus and found that Gap43 expression was downregulated in podocytes of patients with DKD and HG-treated podocytes in vitro. Gap43 knockdown in podocytes promoted podocyte apoptosis, increased migration ability and decreased nephrin expression, while overexpression of Gap43 markedly suppressed HG-induced injury. Moreover, the increased expression and activity of calcineurin (CaN) were also abrogated by overexpression Gap43 in HG. Pretreatment with a typical CaN inhibitor FK506 in Gap43 knockdown podocytes restored the injury. Mechanistically, co-immunoprecipitation experiments suggested that Gap43 could bind to calmodulin (CaM). Pull-down assay further demonstrated that Gap43 and CaM directly interacts with each other via amino acids 30-52 of Gap43 and amino acids 133-197 of CaM. In addition, we also identified Pax5 as potential transcription inhibitor factor mediating Gap43 expression. In conclusion, the study indicated that the Gap43/CaM-CaN pathway may be exploited as a promising therapeutic target for protecting against podocyte injury in high glucose.

Aberrant NAD synthetic flux in podocytes under diabetic conditions and effects of indoleamine 2,3-dioxygenase on promoting de novo NAD synthesis.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme in the kidney. The first step in de novo NAD synthesis is regulated by indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme. Here, we investigated NAD synthetic flux and NAD levels in podocytes under diabetic conditions. We also studied the effects of IDO overexpression on NAD synthetic flux and high glucose (HG)-induced podocyte injury. NAD synthetases in the de novo, Preiss-Handler and salvage pathways were analyzed using in vivo single-nucleus RNA sequencing datasets (GSE131882) of control and diabetic kidney disease (DKD). The mRNA levels of these NAD synthetases were measured in vitro in HG-treated podocytes. The effects of IDO on NAD synthesis were examined by transducing cultured podocytes with an adenovirus encoding IDO, and apoptosis, podocyte markers and mobility were investigated. Cellular transcriptome analysis revealed that control podocytes had relatively low levels of NAD synthetases. In DKD podocytes, de novo NAD synthetase levels were further downregulated. IDO levels were virtually undetectable and did not increase in DKD. In vitro experiments confirmed aberrant de novo NAD synthetic flux and decreased IDO levels in HG-treated podocytes. Overexpression of IDO promoted NAD de novo synthesis, reduced NAD-bypass metabolic enzyme, increased NAD content and recovered podocyte injury markers under diabetic conditions. Taken together, our findings suggest that the de novo NAD synthetic flux is aberrant in DKD, and IDO promotes de novo NAD synthesis and NAD levels, as well as alleviates injury in HG-treated podocytes.

NFAT inhibitor 11R-VIVIT ameliorates mouse renal fibrosis after ischemia-reperfusion-induced acute kidney injury.

Acute kidney injury (AKI) with maladaptive tubular repair leads to renal fibrosis and progresses to chronic kidney disease (CKD). At present, there is no curative drug to interrupt AKI-to-CKD progression. The nuclear factor of the activated T cell (NFAT) family was initially identified as a transcription factor expressed in most immune cells and involved in the transcription of cytokine genes and other genes critical for the immune response. NFAT2 is also expressed in renal tubular epithelial cells (RTECs) and podocytes and plays an important regulatory role in the kidney. In this study, we investigated the renoprotective effect of 11R-VIVIT, a peptide inhibitor of NFAT, on renal fibrosis in the AKI-to-CKD transition and the underlying mechanisms. We first examined human renal biopsy tissues and found that the expression of NFAT2 was significantly increased in RTECs in patients with severe renal fibrosis. We then established a mouse model of AKI-to-CKD transition using bilateral ischemia-reperfusion injury (Bi-IRI). The mice were treated with 11R-VIVIT (5 mg/kg, i.p.) on Days 1, 3, 10, 17 and 24 after Bi-IRI. We showed that the expression of NFAT2 was markedly increased in RTECs in the AKI-to-CKD transition. 11R-VIVIT administration significantly inhibited the nuclear translocation of NFAT2 in RTECs, decreased the levels of serum creatinine and blood urea nitrogen, and attenuated renal tubulointerstitial fibrosis but had no toxic side effects on the heart and liver. In addition, we showed that 11R-VIVIT administration alleviated RTEC apoptosis after Bi-IRI. Consistently, preapplication of 11R-VIVIT (100 nM) and transfection with NFAT2-targeted siRNA markedly suppressed TGFß-induced HK-2 cell apoptosis in vitro. In conclusion, 11R-VIVIT administration inhibits IRI-induced NFAT2 activation and prevents AKI-to-CKD progression. Inhibiting NFAT2 may be a promising new therapeutic strategy for preventing renal fibrosis after IR-AKI.

Integrated analysis of the functions and prognostic values of RNA-binding proteins in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common solid tumor in children. NB treatment has made significant progress; however, given the high degree of heterogeneity, basic research findings and their clinical application to NB still face challenges. Herein, we identify novel prognostic models for NB. METHODS: We obtained RNA expression data of NB and normal nervous tissue from TARGET and GTEx databases and determined the differential expression patterns of RNA binding protein (RBP) genes between normal and cancerous tissues. Lasso regression and Cox regression analyses identified the five most important differentially expressed genes and were used to construct a new prognostic model. The function and prognostic value of these RBPs were systematically studied and the predictive accuracy verified in an independent dataset. RESULTS: In total, 348 differentially expressed RBPs were identified. Of these, 166 were up-regulated and 182 down-regulated RBPs. Two hubs RBPs (CPEB3 and CTU1) were identified as prognostic-related genes and were chosen to build the prognostic risk score models. Multivariate Cox analysis was performed on genes from univariate Cox regression and Lasso regression analysis using proportional hazards regression model. A five gene prognostic model: Risk score = (-0.60901*expCPEB3)+(0.851637*expCTU1) was built. Based on this model, the overall survival of patients in the high-risk subgroup was lower (P = 2.152e-04). The area under the curve (AUC) of the receiver-operator characteristic curve of the prognostic model was 0.720 in the TARGET cohort. There were significant differences in the survival rate of patients in the high and low-risk subgroups in the validation data set GSE85047 (P = 0.1237e-08), with the AUC 0.730. The risk model was also regarded as an independent predictor of prognosis (HR = 1.535, 95% CI = 1.368-1.722, P = 2.69E-13). CONCLUSIONS: This study identified a potential risk model for prognosis in NB using Cox regression analysis. RNA binding proteins (CPEB3 and CTU1) can be used as molecular markers of NB.

Identification of key residues for efficient glucose transport by the hexose transporter CgHxt4 in high sugar fermentation yeast Candida glycerinogenes.

Efficient hexose transporters are essential for the development of industrial yeast strains with high fermentation performance. We previously identified a hexose transporter, CgHxt4, with excellent sugar uptake performance at ultra-high glucose concentrations (200 g/L) in the high sugar fermenting yeast C. glycerinogenes. To understand the working mechanism of this transporter, we constructed 87 mutants and examined their glucose uptake performance. The results revealed that five residues (N321, N322, F325, G426, and P427) are essential for the efficient glucose transport of CgHxt4. Subsequently, we focused our analysis on the roles of N321 and P427. Specifically, N321 and P427 are likely to play a role in glucose coordination and conformational flexibility, respectively. Our results help to expand the application potential of this transporter and provide insights into the working mechanism of yeast hexose transporter. KEY POINTS: • Five residues, transmembrane segments 7 and 10, were found to be essential for CgHxt4. • N321 and P427 are likely to play a role in glucose coordination and conformational flexibility, respectively. • Chimeric CgHxt5.4TM7 significantly enhanced the performance of CgHxt5.

Ultraviolet Irradiation Increased the Concentration of Vitamin D2 and Decreased the Concentration of Ergosterol in Shiitake Mushroom (Lentinus edodes) and Oyster Mushroom (Pleurotus ostreatus) Powder in Ethanol Suspension.

Vitamin D deficiency is a serious global health problem. Edible mushrooms are a good source of vitamin D for human health. The objective of this experiment was to investigate the efficiency of converting its precursor ergosterol to vitamin D2 in shiitake mushroom (Lentinus edodes) and oyster mushroom (Pleurotus ostreatus) powder in ethanol suspension under ultraviolet (UV) irradiation. UV irradiation conditions were optimized for several parameters, such as material form, wavelength, wavelength combination, and exposure time. Under the optimal conditions, UV irradiation increased the concentrations of vitamin D2 from undetectable to 40.59 ± 1.16 µg/g (dw) in dry shiitake mushroom powder and to 677.28 ± 40.42 µg/g (dw) (an approximately 16.69-fold increase) in ethanol suspension. The concentration of vitamin D2 increased from undetectable to 23.71 ± 5.72 µg/g (dw) in the dry oyster mushroom powder upon UV irradiation, whereas UV irradiation increased the concentration to 275.32 ± 48.45 µg/g (dw) (an approximately 11.61-fold increase) in the ethanol suspension. Comparing the effects of varying combinations of wavelengths showed that irradiation with UV-A, UV-C, or a combination of both is more effective than UV-B irradiation. In addition, the increase in vitamin D2 in shiitake mushrooms irradiated by UV-C was time-dependent, that is, dose-dependent. Nevertheless, the increase rates decreased with time. The concentration of ergosterol decreased with the increase in vitamin D2, but ergosterol was only partially converted to vitamin D2, whereas most of the ergosterol was probably UV-degraded. Exposure to ultraviolet light in ethanol suspension offers an effective way to increase the concentration of vitamin D2 and thus improve the nutritional value of edible mushrooms, as well as make them more functional as a source of vitamin D to improve the consumer health.

Identification of Prognostic Genes in Leiomyosarcoma by Gene Co-Expression Network Analysis.

BACKGROUND/AIMS: Leiomyosarcoma (LMS) is a tumor derived from malignant mesenchymal tissue associated with poor prognosis. Determining potential prognostic markers for LMS can provide clues for early diagnosis, recurrence, and treatment. METHODS: RNA sequence data and clinical features of 103 LMS were obtained from the Cancer Genome Atlas (TCGA) database. Application Weighted Gene Co-Expression Network Analysis (WGCNA) was used to construct a free-scale gene co-expression network, to study the interrelationship between its potential modules and clinical features, and to identify hub genes in the module. The hub gene function was verified by an external database. RESULTS: Twenty-four co-expression modules were constructed using WGCNA. A dark red co-expression module was found to be significantly associated with disease recurrence. Functional enrichment analysis and GEPIA and ONCOMINE database analyses demonstrated that hub genes CDK4, CCT2, and MGAT1 may play an important role in LMS recurrence. CONCLUSION: Our study constructed an LMS co-expressing gene module and identified prognostic markers for LMS recurrence detection and treatment.

Stilbene glucoside inhibits the glucuronidation of emodin in rats through the down-regulation of UDP-glucuronosyltransferases 1A8: application to a drug-drug interaction study in Radix Polygoni Multiflori.

ETHNOPHARMACOLOGICAL RELEVANCE: The integrated effects of herbal medicines were the outcome of all of the inherent components. Currently, few studies have focused on the multicomponent interactions in an herbal medicine to elucidate its pharmacological and/or toxicological effects. In this study, an attempt was made to investigate the interaction between stilbene glucosides and the anthraquinones contained in Radix Polygoni Multiflori (RPM) and to explore the interaction's mechanism from the perspective of UDP-glucuronosyltransferase (UGT) regulation. MATERIALS AND METHODS: The extract of RPM was separated into a stilbene glucoside fraction and a emodin fraction. A rapid high-performance liquid chromatography-mass spectrometry method was developed and validated to disclose the influence of stilbene glucoside on the pharmacokinetics of emodin in rats. Drug and Statistics 2.0 was used for the estimation of the pharmacokinetic parameters. Gene expression analysis in liver and intestinal tissues was performed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: The analytical method appeared to be suitable for the analysis of emodin with desirable linearity, accuracy, precision and stability, and the total analysis time was less than 2 min on a short column. Glucuronide of emodin, which is the major metabolite of emodin, was determined after ß-glucuronidase hydrolysis. As the in vivo pharmacokinetic studies had indicated, the AUC, Cmax and T1/2 of emodin were increased after the stilbene glucoside treatment, and the glucuronidation of emodin was significantly inhibited. The mRNA levels from UGT1A8 and UGT1A2 were decreased by stilbene glucoside treatment. In contrast, the expression of UGT1A1, UGT1A6 and UGT1A9 mRNA was increased in the liver following treatment. CONCLUSIONS: The influence of stilbene glucoside on the pharmacokinetics of emodin may be attributed to the inhibition of UGT1A8 mRNA expression. Thus, it is important to extend this research to deepen our understanding of the pharmacological and/or toxicological effects of RPM.

Textural characteristic, antioxidant activity, sugar, organic acid, and phenolic profiles of 10 promising jujube (Ziziphus jujuba Mill.) selections.

UNLABELLED: Ten promising jujubes were analyzed for textural characteristic (hardness), antioxidant activity, sugar, organic acid, and phenolic profiles. Total phenolic content (TP) measured by Folin-Ciocalteu method ranged from 275.6 to 541.8 mg of gallic acid equivalents per 100 g fresh weight (mg GAE/100 g FW). Four sugars (rhamnose, fructose, sucrose, and glucose), 3 organic acids (malic, citric, and succinic acids), and 11 phenolic compounds (gallic acid, protocatechuic acid, cinnamic acid, chlorogenic acid, caffeic acid, ferulic acid, ellagic acid, catechin, epicatechin, rutin, and quercetin) were identified and quantified by high-performance liquid chromatography in jujube fruits. The results showed that ascorbic acid and proanthocyanidins contents were positively correlated with hardness, and antioxidant activity was well correlated with TP content. Among 10 jujube cultivars, Zizyphus jujuba cv. Qingjianmuzao is good for direct consumption containing high levels of total soluble solids, total sugars, fructose, and glucose, while Zizyphus jujuba cv. Jiaxianmuzao could be an important dietary source of natural antioxidants. PRACTICAL APPLICATION: Genotype is the main factor influencing the composition of bioactive compounds in jujubes. Zizyphus jujuba cv. Qingjianmuzao is good for direct consumption, while Zizyphus jujuba cv. Jiaxianmuzao could be an important dietary source of natural antioxidants for prevention of diseases caused by oxidative stress.

Determination of emodin in L-02 cells and cell culture media with liquid chromatography-mass spectrometry: application to a cellular toxicokinetic study.

The emodin-involved hepatotoxicity has been gaining increasing attention. The purpose of the present study was to evaluate the cytotoxicity of emodin on cultured human liver cells (L-02) and predict the possible relation between its cytotoxicity and cellular toxicokinetics. Cell viability and cell damage were assessed by Cell Counting Kit-8 (CCK-8) assay and phase-contrast microscopy, respectively. Cytotoxicity tests demonstrated a concentration- and time-dependent toxic effect of emodin on L-02 cells. Furthermore, emodin at concentration of 30µM led to a significant apoptosis in a time-dependent manner supported by the morphological changes of drug-treated cells. In addition, to elucidate the toxicokinetic characteristics of emodin, a highly sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was employed and validated for detecting the dynamic alteration of emodin in cells and cell culture media. The proposed method appeared to be suitable for the analysis of emodin with desirable linearity (r(2)>0.99), and satisfying precision being less than 8.7%. The range of recoveries of this method was 90.2-101.9%. The preliminary cellular toxicokinetic study revealed a time-dependent intracellular accumulation of emodin, which was consistent with its in vitro toxic effects. These findings confirmed the cytotoxicity of emodin against L-02 cells and displayed the cytotoxic manner of emodin in terms of its cellular uptake and accumulation in L-02 cells.