Transcriptional mechanism of E2F1/TFAP2C/NRF1 in regulating KANK2 gene in nephrotic syndrome.

The mortality rate linked with nephrotic syndrome (NS) is quite high. The renal tubular injury influences the response of NS patients to steroid treatment. KN motif and ankyrin repeat domains 2 (KANK2) regulates actin polymerization, which is required for renal tubular cells to maintain their function. In this study, we found that the levels of KANK2 in patients with NS were considerably lower than those in healthy controls, especially in NS patients with acute kidney injury (AKI). To get a deeper understanding of the KANK2 transcriptional control mechanism, the core promoter region of the KANK2 gene was identified. KANK2 was further found to be positively regulated by E2F Transcription Factor 1 (E2F1), Transcription Factor AP-2 Gamma (TFAP2C), and Nuclear Respiratory Factor 1 (NRF1), both at mRNA and protein levels. Knocking down E2F1, TFAP2C, or NRF1 deformed the cytoskeleton of renal tubular cells and reduced F-actin content. EMSA and ChIP assays confirmed that all three transcription factors could bind to the upstream promoter transcription site of KANK2 to transactivate KANK2 in renal tubular epithelial cells. Our study suggests that E2F1, TFAP2C, and NRF1 play essential roles in regulating the KANK2 transcription, therefore shedding fresh light on the development of putative therapeutic options for the treatment of NS patients.

Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.

Septic acute kidney injury (AKI) is characterized by inflammation. Pyroptosis often occurs during AKI and is associated with the development of septic AKI. This study found that induction of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to a higher level can induce pyroptosis in renal tubular cells. Meanwhile, macrophage migration inhibitory factor (MIF), a subunit of NLRP3 inflammasomes, was essential for IGF2BP1-induced pyroptosis. A putative m6A recognition site was identified at the 3'-UTR region of E2F transcription factor 1 (E2F1) mRNA via bioinformatics analyses and validated using mutation and luciferase experiments. Further actinomycin D (Act D) chase experiments showed that IGF2BP1 stabilized E2F1 mRNA dependent on m6A. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) indicated that E2F1 acted as a transcription factor to promote MIF expression. Thus, IGF2BP1 upregulated MIF through directly upregulating E2F1 expression via m6A modification. Experiments on mice with cecum ligation puncture (CLP) surgery verified the relationships between IGF2BP1, E2F1, and MIF and demonstrated the significance of IGF2BP1 in MIF-associated pyroptosis in vivo. In conclusion, IGF2BP1 was a potent pyroptosis inducer in septic AKI through targeting the MIF component of NLRP3 inflammasomes. Inhibiting IGF2BP1 could be an alternate pyroptosis-based treatment for septic AKI.

Computational detection of a genome instability-derived lncRNA signature for predicting the clinical outcome of lung adenocarcinoma.

Evidence has been emerging of the importance of long non-coding RNAs (lncRNAs) in genome instability. However, no study has established how to classify such lncRNAs linked to genomic instability, and whether that connection poses a therapeutic significance. Here, we established a computational frame derived from mutator hypothesis by combining profiles of lncRNA expression and those of somatic mutations in a tumor genome, and identified 185 candidate lncRNAs associated with genomic instability in lung adenocarcinoma (LUAD). Through further studies, we established a six lncRNA-based signature, which assigned patients to the high- and low-risk groups with different prognosis. Further validation of this signature was performed in a number of separate cohorts of LUAD patients. In addition, the signature was found closely linked to genomic mutation rates in patients, indicating it could be a useful way to quantify genomic instability. In summary, this research offered a novel method by through which more studies may explore the function of lncRNAs and presented a possible new way for detecting biomarkers associated with genomic instability in cancers.

Exosome-mediated pyroptosis of miR-93-TXNIP-NLRP3 leads to functional difference between M1 and M2 macrophages in sepsis-induced acute kidney injury.

Sepsis is a systemic inflammatory response syndrome caused by infection, resulting in organ dysfunction. Sepsis-induced acute kidney injury (AKI) is one of the most common potential complications. Increasing reports have shown that M1 and M2 macrophages both take part in the progress of AKI by influencing the level of inflammatory factors and the cell death, including pyroptosis. However, whether M1 and M2 macrophages regulate AKI by secreting exosome remains unknown. In the present study, we isolated the exosomes from M1 and M2 macrophages and used Western blot and enzyme-linked immunosorbent assay (ELISA) to investigate the effect of M1 and M2 exosomes on cell pyroptosis. miRNA sequencing was used to identify the different miRNA in M1 and M2 exosomes. Luciferase reporter assay was used to verify the target gene of miRNA. We confirmed that exosomes excreted by macrophages regulated cell pyroptosis in vitro by using Western blot and ELISA. miRNA sequencing revealed the differentially expressed level of miRNAs in M1 and M2 exosomes, among which miR-93-5p was involved in the regulation of pyroptosis. By using bioinformatics predictions and luciferase reporter assay, we found that thioredoxin-interacting protein (TXNIP) was a direct target of miR-93-5p. Further in vitro and in vivo experiments indicated that exosomal miR-93-5p regulated the TXNIP directly to influence the pyroptosis in renal epithelial cells, which explained the functional difference between different phenotypes of macrophages. This study might provide new targets for the treatment of sepsis-induced AKI.

Cervicovaginal microbiota dysbiosis correlates with HPV persistent infection.

HPV persistent infection is a main event leading to the development of cervical intraepithelial neoplasia and cervical cancer. Earlier to distinguish HPV persistent and transient infection is meaningful but the methods are limited. This study used 16S rDNA sequencing to determine the cervicovaginal microbiota of HPV persistent infection, transient infection and health women. Sequences analysis was performed and according to subsequent statistical analysis, the structure of cervicovaginal microbiota of healthy and transient infection individuals is relatively single, Firmicutes occupy the main composition. However, that of the HPV persistent infection presented a complicated trend and the abundance of Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria was higher. The significance p-values of the average species abundance of Firmicutes, Proteobacteria and Bacteroides between HPV persistent and transient infection groups were 0.003, 0.018 and 0.005, respectively. The study also found 36 biomarkers of cervicovaginal microbiota dysbiosis for LDA score>4 among different groups. At genus level, Prevotella, Sphingomonas and Anaerococcus correlated with HPV persistent infection. At species level, Lactobacillus iners correlated with HPV transient infection. Besides, local immune microenvironment was changed with cervicovaginal microbiota dysbiosis. Interleukin-6 and TNF-α were significantly upregulated in cervical secretions from HPV persistent infection compared with those from transient infection and healthy women. Peripheral blood Regulatory T cells and myeloid-derived suppressor cells in patients with HPV persistent infection were also significantly increased. In conclusion, this study identified cervicovaginal microbiota dysbiosis closely related to HPV persistent infection, which provided a new idea and method for the prevention of cervical cancer.

Long non-coding RNA MALAT1 targeting STING transcription promotes bronchopulmonary dysplasia through regulation of CREB.

Bronchopulmonary dysplasia (BPD) is a severe complication of preterm infants characterized by increased alveolarization and inflammation. Premature exposure to hyperoxia is believed to be a key contributor to the pathogenesis of BPD. No effective preventive or therapeutic agents have been created. Stimulator of interferon gene (STING) is associated with inflammation and apoptosis in various lung diseases. Long non-coding RNA MALAT1 has been reported to be involved in BPD. However, how MALAT1 regulates STING expression remains unknown. In this study, we assessed that STING and MALAT1 were up-regulated in the lung tissue from BPD neonates, hyperoxia-based rat models and lung epithelial cell lines. Then, using the flow cytometry and cell proliferation assay, we found that down-regulating of STING or MALAT1 inhibited the apoptosis and promoted the proliferation of hyperoxia-treated cells. Subsequently, qRT-PCR, Western blotting and dual-luciferase reporter assays showed that suppressing MALAT1 decreased the expression and promoter activity of STING. Moreover, transcription factor CREB showed its regulatory role in the transcription of STING via a chromatin immunoprecipitation. In conclusion, MALAT1 interacts with CREB to regulate STING transcription in BPD neonates. STING, CREB and MALAT1 may be promising therapeutic targets in the prevention and treatment of BPD.

Hypermethylation of mismatch repair gene hMSH2 associates with platinum-resistant disease in epithelial ovarian cancer.

PURPOSE: One major reason of the high mortality of epithelial ovarian cancer (EOC) is due to platinum-based chemotherapy resistance. Aberrant DNA methylation may be a potential mechanism underlying the development of platinum resistance in EOC. The purpose of this study is to discover potential aberrant DNA methylation that contributes to drug resistance. METHODS: By initially screening of 16 platinum-sensitive/resistant samples from EOC patients with reduced representation bisulfite sequencing (RRBS), the upstream region of the hMSH2 gene was discovered hypermethylated in the platinum-resistant group. The effect of hMSH2 methylation on the cellular response to cisplatin was explored by demethylation and knockdown assays in ovarian cancer cell line A2780. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was employed to examine the methylation levels of hMSH2 upstream region in additional 40 EOC patient samples. RT-qPCR and IHC assay was used to detect the hMSH2 mRNA and protein expression in extended 150 patients. RESULTS: RRBS assay discovered an upstream region from - 1193 to - 1125 of hMSH2 was significant hypermethylated in resistant EOC patients (P = 1.06 × 10-14). In vitro analysis demonstrated that global demethylation increased cisplatin sensitivity along with a higher expression of the hMSH2 mRNA and protein. Knockdown hMSH2 reduced the cell sensitivity to cisplatin. MALDI-TOF mass spectrometry assay validated the strong association of hypermethylation of hMSH2 upstream region with platinum resistance. Spearman's correlation analysis revealed a significantly negative connection between methylation level of hMSH2 upstream region and its expression. The Kaplan-Meier analyses showed the high methylation of hMSH2 promoter region, and its low expressions are associated with worse survival. In multivariable models, hMSH2 low expression was an independent factor predicting poor outcome (P = 0.03, HR = 1.91, 95%CI = 1.85-2.31). CONCLUSION: The hypermethylation of hMSH2 upstream region is associated with platinum resistant in EOC, and low expression of hMSH2 may be an index for the poor prognosis.

Methylation status of ORMDL3 regulates cytokine production and p-ERK/MMP9 pathway expression.

Orosomucoid like-3 (ORMDL3) has been identified to be associated with the development of asthma according to previous studies. However, the definite role of ORMDL3 in the pathogenesis of asthma remains unclear. In this study, we found ORMDL3 was highly expressed in PBMC specimens from childhood asthma patients. Cytokines production and p-ERK/MMP-9 pathway expression was also increased in childhood asthma patients compared with controls. In addition, ORMDL3 overexpression induced IL-6 and IL-8 release and activated p-ERK/MMP-9 pathway in vitro. Increased ORMDL3 expression was observed after treated with 5-Aza-CdR. 5-Aza-CdR decreased the percentage of the CpG island in the ORMDL3 promoter region and increased its promoter activity. In addition, 5-Aza-CdR significantly increased IL-6 and IL-8 levels in NHBE cells while there was no obvious alteration after knocking down ORMDL3. Knockdown of ORMDL3 also significantly decreased the expression of p-ERK/MMP-9 pathway in the presence or absence of 5-Aza-CdR. In conclusion, our study provided novel evidence for the association between ORMDL3 and asthma-associated cytokines. Moreover, DNA methylation plays an important role in ORMDL3-mediated increased IL-6 and IL-8 levels and p-ERK/MMP-9 pathway expression.

Performance Evaluation of a Newly Developed Chemiluminescent Assay Kit for Detection of Neuron-Specific Eno.

BACKGROUND: To evaluate the performance of a chemiluminescence detection reagent for Neuron-specific enolase (NSE). METHODS: Based on the "Guiding principles on performance analysis of diagnostic, reagents in vitro" and the Clinical and Laboratory Standards Institute (CLSI) Guidelines, performance of the CLIA NSE kit was evaluated, including the detection limit, linear range, reportable range, accuracy, precision, cross reactivity, interference factors, Hook effect, and method comparison. RESULTS: The detection limit of the reagent was 0.05 ng/mL. The linear range of the reagent was 0.05 ng/mL - 400 ng/mL. The reagent can be reported as 0.05 ng/mL - 2,500 ng/mL. The recovery rate ranged from 94.95% to 105.12%. The CV of the reagent of the intra-assay was 3.8% - 5.7% and inter-batch was 3.6%, which meets the requirements. The common interference factors such as the blood fat, jaundice, and rheumatoid factor did not affect the quantitative accuracy of the reagent, but hemolysis resulted in higher readings. Cross-reactions were not observed when incubating with major interfering tumor markers; therefore, the kit was highly specific for NSE. The HOOK effect was not observed when the NSE content reached 20,000 ng/mL in samples. The coincidence rate of the reagent and Roche's products reached 94.81% and the correlation r reached 0.968. CONCLUSIONS: The performance of the NSE CLIA reagent was acceptable in all evaluated parameters, meeting requirements for clinical application.

SIRT6 Depletion Sensitizes Human Hepatoma Cells to Chemotherapeutics by Downregulating MDR1 Expression.

Multidrug resistance (MDR) due to overexpression of MDR1 is a major obstacle that hinders the treatment of hepatocellular carcinoma (HCC). In this study, we explored the function and underlying molecular mechanism of SIRT6 in MDR of HCC. Chemotherapeutic agents (doxorubicin, cisplatin, and sorafenib) treatment increased SIRT6 mRNA and protein level in two HCC cell lines in a dose-dependent manner. SIRT6 depletion resulted in decreased cell viability and increased apoptosis in HCC cells treated with chemotherapeutic agents. Mechanistically, SIRT6 depletion reduced MDR1 transcription by targeting its promoter in HCC cells treated with chemotherapeutic agents. Consistently, the protein level of MDR1 was also reduced in SIRT6-depleted HCC cells. Further studies indicated that SIRT6 depletion may suppress CCAAT/enhancer binding protein ß (C/EBPß), to act as a transcriptional activator of MDR1 in HCC cells treated with chemotherapeutic agents. Importantly, forced expression of MDR1 could attenuate the apoptosis induced by chemotherapeutic agents in SIRT6-depleted cells. Taken together, these results indicated SIRT6 depletion enhanced chemosensitivity of human hepatoma cells by downregulating MDR1 expression through suppressing C/EBPß. SIRT6 may serve as a novel target to enhance chemosensitivity in HCC cells.

Cigarette smoke extract induces apoptosis of rat alveolar Type II cells via the PLTP/TGF-ß1/Smad2 pathway.

Apoptosis of alveolar epithelial cells has been implicated in the pathogenesis of acute lung injury. Phospholipid transfer protein (PLTP) may play a role in apoptosis. In the present study, the effect of the novel function of PLTP in cigarette smoke extract (CSE)-induced apoptosis of alveolar epithelial cells and the possible mechanism were examined. Male Wistar rats were exposed to air and cigarette smoke (n=10/exposure) for 6h/day on 3 consecutive days, then the lungs were sectioned and examined. To investigate effects on alveolar epithelial cells, rat alveolar epithelial cells (RLE-6TN) were treated with different concentrations of CSE for various times. siRNA for PLTP was transfected into cells and an inhibitor of the transforming growth factor-ß1 (TGF-ß1) type I receptor was administered prior to CSE exposure. Apoptosis was measured, and mRNA expression of PLTP and TGF-ß1 and protein levels of PLTP, TGF-ß1, p-Smad2 and cleaved caspase-3 were analyzed. The results showed that apoptosis, as well as expression of PLTP, TGF-ß1, p-Smad2 and cleaved caspase-3 were all significantly increased after CSE stimulation (P<0.05). Furthermore, the expression of TGF-ß1, p-Smad2 and cleaved caspase-3 induced by CSE could be partly abrogated by knockdown of PLTP. The expression of PLTP showed no significant change as a result of TGF-ß1 receptor inhibition, while cleaved caspase-3 showed a remarkable reduction. PLTP may act as an upstream signal molecule of the TGF-ß1/Smad2 pathway and is likely to be involved in CSE-induced apoptosis of alveolar epithelial cells.

Numerical solutions of ideal quantum gas dynamical flows governed by semiclassical ellipsoidal-statistical distribution.

The ideal quantum gas dynamics as manifested by the semiclassical ellipsoidal-statistical (ES) equilibrium distribution derived in Wu et al. (Wu et al. 2012 Proc. R. Soc. A468, 1799-1823 (doi:10.1098/rspa.2011.0673)) is numerically studied for particles of three statistics. This anisotropic ES equilibrium distribution was derived using the maximum entropy principle and conserves the mass, momentum and energy, but differs from the standard Fermi-Dirac or Bose-Einstein distribution. The present numerical method combines the discrete velocity (or momentum) ordinate method in momentum space and the high-resolution shock-capturing method in physical space. A decoding procedure to obtain the necessary parameters for determining the ES distribution is also devised. Computations of two-dimensional Riemann problems are presented, and various contours of the quantities unique to this ES model are illustrated. The main flow features, such as shock waves, expansion waves and slip lines and their complex nonlinear interactions, are depicted and found to be consistent with existing calculations for a classical gas.

The profile in polyphenols and volatile compounds in alcoholic beverages from different cultivars of mulberry.

UNLABELLED: Mulberry fruits have different chemical compositions because of cultivar variation. In this study, polyphenol profile and volatile composition of alcoholic beverages produced from different mulberries of Da 10, Hongguo 2 hao, and Hongguo 1 hao were investigated. Statistically significant differences were detected in the chemical composition of the analyzed alcoholic beverage samples. The beverage of Da 10 possessed the highest content of total phenols, total flavonoids, and total anthocyanins, which was determined by spectrophotometry analysis, contrarily Hongguo 1 hao beverage showing the lowest. Phenolic acids and flavonols were analyzed by high performance liquid chromatography analysis. Protocatechuic acid was the main phenolic acid in all analyzed alcoholic beverage samples, and its concentration ranged from 8.20 mg L⁻¹ (Hongguo 1 hao beverage) to 29.98 mg L⁻¹ (Da 10 beverage). High level of flavonols was found in both Da 10 and Hongguo 2 hao beverage samples, whereas Hongguo 1 hao beverage sample was characterized with lower level of flavonols. Furthermore, volatile compounds were analyzed by solid phase microextraction-gas chromatography mass spectrometry analysis. The amount of esters varied widely among the 3 alcoholic beverage samples. The concentration of aliphatic alcohols in Hongguo 2 hao and 1 hao beverage samples was higher than Da 10 sample, whereas Da 10 beverage sample contained more aromatic alcohols than the others. Hongguo 1 beverage sample showed the highest concentration of acetic acid. This study demonstrated that quantitative differences of phenolic compounds and volatile compounds among cultivars were significant, which highly affected the quality of alcoholic beverage from mulberry. PRACTICAL APPLICATION: Mulberry fruits had different chemical compositions because of cultivar variation. The analyzed Da 10 cultivar of mulberry appeared to be a good raw material for alcoholic beverage-making because of its high level of total sugar and acidity. This study demonstrated that alcoholic beverages from mulberry contain high amounts of polyphenols. These bioactive compounds may act synergistically to promote health, and reduce risk factors of chronic diseases.