Cytosolic DNA initiates a vicious circle of aging-related endothelial inflammation and mitochondrial dysfunction via STING: the inhibitory effect of Cilostazol.

Endothelial senescence, aging-related inflammation, and mitochondrial dysfunction are prominent features of vascular aging and contribute to the development of aging-associated vascular disease. Accumulating evidence indicates that DNA damage occurs in aging vascular cells, especially in endothelial cells (ECs). However, the mechanism of EC senescence has not been completely elucidated, and so far, there is no specific drug in the clinic to treat EC senescence and vascular aging. Here we show that various aging stimuli induce nuclear DNA and mitochondrial damage in ECs, thus facilitating the release of cytoplasmic free DNA (cfDNA), which activates the DNA-sensing adapter protein STING. STING activation led to a senescence-associated secretory phenotype (SASP), thereby releasing pro-aging cytokines and cfDNA to further exacerbate mitochondrial damage and EC senescence, thus forming a vicious circle, all of which can be suppressed by STING knockdown or inhibition. Using next-generation RNA sequencing, we demonstrate that STING activation stimulates, whereas STING inhibition disrupts pathways associated with cell senescence and SASP. In vivo studies unravel that endothelial-specific Sting deficiency alleviates aging-related endothelial inflammation and mitochondrial dysfunction and prevents the development of atherosclerosis in mice. By screening FDA-approved vasoprotective drugs, we identified Cilostazol as a new STING inhibitor that attenuates aging-related endothelial inflammation both in vitro and in vivo. We demonstrated that Cilostazol significantly inhibited STING translocation from the ER to the Golgi apparatus during STING activation by targeting S162 and S243 residues of STING. These results disclose the deleterious effects of a cfDNA-STING-SASP-cfDNA vicious circle on EC senescence and atherogenesis and suggest that the STING pathway is a promising therapeutic target for vascular aging-related diseases. A proposed model illustrates the central role of STING in mediating a vicious circle of cfDNA-STING-SASP-cfDNA to aggravate age-related endothelial inflammation and mitochondrial damage.

Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/ß-catenin signaling in obstructed kidneys in vivo.

Follistatin (FS)-like 1 (FSTL1) is a member of the FS-SPARC (secreted protein, acidic and rich in cysteine) family of secreted and extracellular matrix proteins. The functions of FSTL1 have been studied in heart and lung injury as well as in wound healing; however, the role of FSTL1 in the kidney is largely unknown. Here, we show using single-cell RNA-Seq that Fstl1 was enriched in stromal cells in obstructed mouse kidneys. In addition, immunofluorescence demonstrated that FSTL1 expression was induced in fibroblasts during kidney fibrogenesis in mice and human patients. We demonstrate that FSTL1 overexpression increased renal fibrosis and activated the Wnt/ß-catenin signaling pathway, known to promote kidney fibrosis, but not the transforming growth factor ß (TGF-ß), Notch, Hedgehog, or Yes-associated protein (YAP) signaling pathways in obstructed mouse kidneys, whereas inhibition of FSTL1 lowered Wnt/ß-catenin signaling. Importantly, we show that FSTL1 interacted with Wnt ligands and the Frizzled (FZD) receptors but not the coreceptor lipoprotein receptor-related protein 6 (LRP6). Specifically, we found FSTL1 interacted with Wnt3a through its extracellular calcium-binding (EC) domain and von Willebrand factor type C-like (VWC) domain, and with FZD4 through its EC domain. Furthermore, we show that FSTL1 increased the association of Wnt3a with FZD4 and promoted Wnt/ß-catenin signaling and fibrogenesis. The EC domain interacting with both Wnt3a and FZD4 also enhanced Wnt3a signaling. Therefore, we conclude that FSTL1 is a novel extracellular enhancer of the Wnt/ß-catenin pathway.

SGLT2 inhibitors improve kidney function and morphology by regulating renal metabolic reprogramming in mice with diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD) worldwide. SGLT2 inhibitors are clinically effective in halting DKD progression. However, the underlying mechanisms remain unclear. The serum and kidneys of mice with DKD were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomic and proteomic analyses. Three groups were established: placebo-treated littermate db/m mice, placebo-treated db/db mice and EMPA-treated db/db mice. Empagliflozin (EMPA) and placebo (10 mg/kg/d) were administered for 12 weeks. EMPA treatment decreased Cys-C and urinary albumin excretion compared with placebo by 78.60% and 57.12%, respectively (p < 0.001 in all cases). Renal glomerular area, interstitial fibrosis and glomerulosclerosis were decreased by 16.47%, 68.50% and 62.82%, respectively (p < 0.05 in all cases). Multi-omic analysis revealed that EMPA treatment altered the protein and metabolic profiles in the db/db group, including 32 renal proteins, 51 serum proteins, 94 renal metabolites and 37 serum metabolites. Five EMPA-related metabolic pathways were identified by integrating proteomic and metabolomic analyses, which are involved in renal purine metabolism; pyrimidine metabolism; tryptophan metabolism; nicotinate and nicotinamide metabolism, and glycine, serine and threonine metabolism in serum. In conclusion, this study demonstrated metabolic reprogramming in mice with DKD. EMPA treatment improved kidney function and morphology by regulating metabolic reprogramming, including regulation of renal reductive stress, alleviation of mitochondrial dysfunction and reduction in renal oxidative stress reaction.

Anthocyanin improves kidney function in diabetic kidney disease by regulating amino acid metabolism.

BACKGROUND: Diabetic kidney disease (DKD) is among the most important causes for chronic kidney disease. Anthocyanins (ANT) are polyphenolic compounds present in various food and play an important role in ameliorating hyperglycemia and insulin sensitivity. However, the effects of ANT in DKD are still poorly understood. This study aimed to investigate the effect of ANT (cyanidin-3-O-glucoside [C3G]) on the renal function of DKD, and whether the anti-DKD effect of ANT is related to metabolic pathways. METHODS: To explore the role of ANT in DKD, we performed the examination of blood glucose, renal function, and histopathology. As for the mechanism, we designed the label-free quantification proteomics and nontargeted metabolomics analysis for kidney and serum. Subsequently, we revealed the anti-DKD effect of ANT through the bioinformatic analysis. RESULTS: We showed that the fasting blood glucose level (- 6.1 mmol/L, P = 0.037), perimeter of glomerular lesions (- 24.1 µm, P = 0.030), fibrosis score of glomerular (- 8.8%, P = 0.002), and kidney function (Cystatin C: - 701.4 pg/mL, P = 0.043; urine creatinine: - 701.4 mmol/L, P = 0.032) were significantly alleviated in DKD mice after ANT treatment compared to untreated in the 20th week. Further, proteins and metabolites in the kidneys of DKD mice were observed to be dramatically altered due to changes in amino acid metabolism with ANT treatment; mainly, taurine and hypotaurine metabolism pathway was upregulated (P = 0.0001, t value = 5.97). Furthermore, upregulated tryptophan metabolism (P < 0.0001, t value = 5.94) and tyrosine metabolism (P = 0.0037, t value = 2.91) pathways had effects on serum of DKD mice as responsed ANT regulating. CONCLUSIONS: Our results suggested that prevention of the progression of DKD by ANT could be related to the regulation of amino acid metabolism. The use of dietary ANT may be one of the dietary strategies to prevent and treat DKD.

Molecular subtypes based on DNA methylation predict prognosis in lung squamous cell carcinoma.

BACKGROUND: Due to tumor heterogeneity, the diagnosis, treatment, and prognosis of patients with lung squamous cell carcinoma (LUSC) are difficult. DNA methylation is an important regulator of gene expression, which may help the diagnosis and therapy of patients with LUSC. METHODS: In this study, we collected the clinical information of LUSC patients in the Cancer Genome Atlas (TCGA) database and the relevant methylated sequences of the University of California Santa Cruz (UCSC) database to construct methylated subtypes and performed prognostic analysis. RESULTS: Nine hundred sixty-five potential independent prognosis methylation sites were finally identified and the genes were identified. Based on consensus clustering analysis, seven subtypes were identified by using 965 CpG sites and corresponding survival curves were plotted. The prognostic analysis model was constructed according to the methylation sites' information of the subtype with the best prognosis. Internal and external verifications were used to evaluate the prediction model. CONCLUSIONS: Models based on differences in DNA methylation levels may help to classify the molecular subtypes of LUSC patients, and provide more individualized treatment recommendations and prognostic assessments for different clinical subtypes. GNAS, FZD2, FZD10 are the core three genes that may be related to the prognosis of LUSC patients.

LncRNA HOTTIP inhibits cell pyroptosis by targeting miR-148a-3p/AKT2 axis in ovarian cancer.

Long noncoding RNA HOTTIP is a crucial regulator in multiple types of cancer, including ovarian cancer (OC). However, the biological roles and underlying mechanisms of HOTTIP in OC have rarely been studied. Hence, this study aimed to investigate the functional correlation between HOTTIP and pyroptosis in OC progression. The expression of HOTTIP in OC tissues and cell lines was characterized by quantitative real-time PCR. Cell proliferation was evaluated using Cell Counting Kit-8 and clone formation assays. Western blot was performed to quantify protein levels. A dual-luciferase reporter assay was used to analyze the molecular interaction among HOTTIP, miR-148a-3p, and AKT2. The expression of HOTTIP was significantly upregulated in OC tissue samples and cell lines. The silencing of HOTTIP led to the inhibition of cell proliferation and NLRP1 inflammasome-mediated pyroptosis. In addition, HOTTIP increased AKT2 expression by negatively regulating miR-148a-3p and then inhibited ASK1/JNK signaling. Further rescue experiments revealed that downregulation of miR-148a-3p and overexpression of AKT2 obviously diminished the effects of HOTTIP downregulation in OC cells. Thus, our study elucidated a novel pyroptosis-related mechanism by which HOTTIP participated in OC progression, which might provide a theoretical reference for clinical treatment.

RGMb protects against acute kidney injury by inhibiting tubular cell necroptosis via an MLKL-dependent mechanism.

Tubular cell necrosis is a key histological feature of acute kidney injury (AKI). Necroptosis is a type of programed necrosis, which is executed by mixed lineage kinase domain-like protein (MLKL) upon its binding to the plasma membrane. Emerging evidence indicates that necroptosis plays a critical role in the development of AKI. However, it is unclear whether renal tubular cells undergo necroptosis in vivo and how the necroptotic pathway is regulated during AKI. Repulsive guidance molecule (RGM)-b is a member of the RGM family. Our previous study demonstrated that RGMb is highly expressed in kidney tubular epithelial cells, but its biological role in the kidney has not been well characterized. In the present study, we found that RGMb reduced membrane-associated MLKL levels and inhibited necroptosis in cultured cells. During ischemia/reperfusion injury (IRI) or oxalate nephropathy, MLKL was induced to express on the apical membrane of proximal tubular (PT) cells. Specific knockout of Rgmb in tubular cells (Rgmb cKO) increased MLKL expression at the apical membrane of PT cells and induced more tubular cell death and more severe renal dysfunction compared with wild-type mice. Treatment with the necroptosis inhibitor Necrostatin-1 or GSK'963 reduced MLKL expression on the apical membrane of PT cells and ameliorated renal function impairment after IRI in both wild-type and Rgmb cKO mice. Taken together, our results suggest that proximal tubular cell necroptosis plays an important role in AKI, and that RGMb protects against AKI by inhibiting MLKL membrane association and necroptosis in proximal tubular cells.

Tubule-Specific Mst1/2 Deficiency Induces CKD via YAP and Non-YAP Mechanisms.

BACKGROUND: The serine/threonine kinases MST1 and MST2 are core components of the Hippo pathway, which has been found to be critically involved in embryonic kidney development. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the pathway's main effectors. However, the biologic functions of the Hippo/YAP pathway in adult kidneys are not well understood, and the functional role of MST1 and MST2 in the kidney has not been studied. METHODS: We used immunohistochemistry to examine expression in mouse kidneys of MST1 and MST2, homologs of Hippo in Drosophila. We generated mice with tubule-specific double knockout of Mst1 and Mst2 or triple knockout of Mst1, Mst2, and Yap. PCR array and mouse inner medullary collecting duct cells were used to identify the primary target of Mst1/Mst2 deficiency. RESULTS: MST1 and MST2 were predominantly expressed in the tubular epithelial cells of adult kidneys. Deletion of Mst1/Mst2 in renal tubules increased activity of YAP but not TAZ. The kidneys of mutant mice showed progressive inflammation, tubular and glomerular damage, fibrosis, and functional impairment; these phenotypes were largely rescued by deletion of Yap in renal tubules. TNF-α expression was induced via both YAP-dependent and YAP-independent mechanisms, and TNF-α and YAP amplified the signaling activities of each other in the tubules of kidneys with double knockout of Mst1/Mst2. CONCLUSIONS: Our findings show that tubular Mst1/Mst2 deficiency leads to CKD through both the YAP and non-YAP pathways and that tubular YAP activation induces renal fibrosis. The pathogenesis seems to involve the reciprocal stimulation of TNF-α and YAP signaling activities.

Lethal (3) malignant brain tumor-like 2 (L3MBTL2) protein protects against kidney injury by inhibiting the DNA damage-p53-apoptosis pathway in renal tubular cells.

DNA damage contributes to renal tubular cell death during kidney injury, but how DNA damage in tubular cells is regulated is not fully understood. Lethal (3) malignant brain tumor-like 2 (L3MBTL2), a novel polycomb group protein, has been implicated in regulating chromatin architecture. However, the biological functions of L3MBTL2 are largely undefined. Here we found that L3MBTL2 was expressed in the nuclei of renal tubular epithelial cells in mice. Ablation of L3mbtl2 in renal tubular cells resulted in increases in nuclear DNA damage, p53 activation, apoptosis, tubular injury and kidney dysfunction after cisplatin treatment or unilateral ureteral obstruction. In vitro, inhibition of L3MBTL2 sequentially promoted histone γH2AX expression, p53 activation and apoptosis in cisplatin-treated mouse proximal tubular TKPTS cells. Inhibition of p53 activity attenuated the apoptosis induced by L3mbtl2 deficiency after cisplatin treatment both in vivo and in vitro. Intriguingly, unlike other polycomb proteins, L3MBTL2 was not recruited to DNA damage sites, but instead increased nuclear chromatin density and reduced initial DNA damage load. Thus, L3MBTL2 plays a protective role in kidney injury, in part by inhibiting the DNA damage-p53-apoptosis pathway.

Enhanced glucose homeostasis via Clostridium symbiosum-mediated glucagon-like peptide 1 inhibition of hepatic gluconeogenesis in mid-intestinal bypass surgery.

BACKGROUND: The small intestine is known to play a crucial role in the development and remission of diabetes mellitus (DM). However, the exact mechanism by which mid-small intestinal bypass improves glucose metabolism in diabetic rats is not fully understood. AIM: To elucidate the mechanisms by which mid-small intestinal bypass improves glucose metabolism. METHODS: Streptozotocin (STZ) was used to induce DM in Sprague-Dawley (SD) rats at a dose of 60 mg/kg. The rats were then randomly divided into two groups: The mid-small intestine bypass (MSIB) group and the sham group (underwent switch laparotomy). Following a 6-wk recovery period post-surgery, the rats underwent various assessments, including metabolic parameter testing, analysis of liver glycogen levels, measurement of key gluconeogenic enzyme activity, characterization of the gut microbiota composition, evaluation of hormone levels, determination of bile acid concentrations, and assessment of the expression of the intestinal receptors Takeda G protein-coupled receptor 5 and farnesoid X receptor. RESULTS: The MSIB group of rats demonstrated improved glucose metabolism and lipid metabolism, along with increased hepatic glycogen content. Furthermore, there was a decrease in the expression of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 and glucose-6-phosphatase. Importantly, the MSIB group exhibited a substantial increase in the abundances of intestinal Lactobacillus, Clostridium symbiosum, Ruminococcus gnavus, and Bilophila. Moreover, higher levels of secondary bile acids, such as intestinal lithocholic acid, were observed in this group. Remarkably, the changes in the gut microbiota showed a significant correlation with the expression of key gluconeogenic enzymes and glucagon-like peptide 1 (GLP-1) at 6 wk postoperatively, highlighting their potential role in glucose regulation. These findings highlight the beneficial effects of mid-small intestine bypass on glucose metabolism and the associated modulation of the gut microbiota. CONCLUSION: The findings of this study demonstrate that the introduction of postoperative intestinal Clostridium symbiosum in the mid-small intestine contributes to the enhancement of glucose metabolism in nonobese diabetic rats. This improvement is attributed to the increased inhibition of hepatic gluconeogenesis mediated by GLP-1, resulting in a favorable modulation of glucose homeostasis.

Differences in metabolic improvement after metabolic surgery are linked to the gut microbiota in non-obese diabetic rats.

BACKGROUND: Different metabolic/bariatric surgery approaches vary in their effect on weight loss and glucose levels, although the underlying mechanism is unclear. Studies have demonstrated that the gut microbiota might be an important mechanism of improved metabolism after metabolic/bariatric surgery. AIM: To investigate the relationship between the improvement in metabolic disturbances and the changes in gut microbiota after gastric or intestinal bypass. METHODS: We performed sleeve gastrectomy (SG), distal small intestine bypass (DSIB) or sham surgery in nonobese rats with diabetes induced by 60 mg/kg streptozotocin (STZ-DM). RESULTS: The group comparisons revealed that both SG and DSIB induced a reduction in body weight and significant improvements in glucose and lipid metabolism in the STZ-DM rats. Furthermore, DSIB exhibited a stronger glucose-lowering and lipid-reducing effect on STZ-DM rats than SG. 16S ribosomal RNA gene sequencing revealed the gut abundance of some Lactobacillus spp. increased in both the SG and DSIB groups after surgery. However, the DSIB group exhibited a more pronounced increase in the gut abundance of Lactobacillus spp. compared to the SG group, with more Lactobacillus spp. types increased in the gut. CONCLUSION: The gut abundance of Lactobacillus was significantly correlated with the improvement in glycolipid metabolism and the change in serum fibroblast growth factor 21 levels.

The role of microbial metabolites in diabetic kidney disease.

Background: Growing evidence suggests a complex bidirectional interaction between gut microbes, gut-derived microbial metabolites, and diabetic kidney disease (DKD), known as the "gut-kidney axis" theory. The present study aimed to characterize the role of microbial metabolites in DKD. Methods: Six-week-old db/db and littermate db/m mice were raised to 20 weeks old. The serum, urine, feces, liver, perinephric fat, and kidney were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomic analyses. Results: The db/db mice showed obvious pathological changes and worse renal functions than db/m mice. Indoleacetaldehyde (IAld) and 5-hydroxy-l-tryptophan (5-HTP) in kidney samples, and serotonin (5-HT) in fecal samples were increased in the db/db group. Phosphatidylcholine (PC), phosphatidate (PA), and 1-acylglycerophosphocholine (lysoPC) were decreased in liver and serum samples of the db/db group, while PC and lysoPC were decreased in kidney and perinephric fat samples. Suggested metabolomic homeostasis was disrupted in DKD mice, especially glycerophospholipid and tryptophan metabolism, which are closely related to the gut microbiome. Conclusions: Our findings reveal the perturbation of gut microbial metabolism in db/db mice with DKD, which may be useful for building a bridge between the gut microbiota and the progression of DKD and provide a theoretical basis for the intestinal treatment of DKD.

Plasma adsorption in refractory chronic gouty arthritis flare: A case report.

Background: Along with uric acid, which is the primary driving factor of gout, downstream inflammatory mediators have been shown to be involved in the pathogenesis of gouty arthritis flares. Extracorporeal haemadsorption is an emerging technology for the treatment of dysregulated inflammatory states by effectively removing cytokines from the bloodstream. Whether haemadsorption was effective in refractory gout flares has not been reported in the literature. Case summary: We report the case of a 52-year-old male who presented with refractory gouty arthropathy for 30 years. His uric acid levels were poorly controlled due to poor diet and treatment compliance. Tophi were found to have precipitated in multiple joints and subcutaneous tissue. In the last 2 years, his incidents of gouty flares had become more frequent, and resistant to the medications, including colchicine, allopurinol, febuxostat, glucocorticoids, and NSAID analgesics. He had experienced a triad of chills, high fever and arthritis for the past 2 weeks. Therefore, he took 2 mg colchicine twice daily for 2 weeks with no improvement in his pain. Proinflammatory cytokines, such as interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α), were found to be remarkably elevated. Given that conventional treatment was unsuccessful, we tried to employ plasma adsorption (PA) to remove inflammatory cytokines. After 4 sessions, symptoms, such as fever, joint swelling and pain, were greatly improved. Meanwhile, the levels of proinflammatory factors such as IL-6 and TNF-α were found to be decreased, while the anti-inflammatory factor IL-10 remained the same during the course. He was followed up for 8 months and arthritis have flared up twice in response to a high-purine diet. Conclusion: Our study suggests that plasma adsorption (PA) may be a promising and feasible treatment for refractory gout when conventional treatments are unsatisfactory or contraindicated. However, more clinical trials are needed to verify the efficacy and safety of the treatment. Core tip: Chronic gouty arthritis flares are refractory to conventional treatment, such as uric acid-lowering drugs and NSAID analgesics. Due to the involvement of inflammatory cytokines, plasma adsorption was employed to alleviate flares by removing inflammatory mediators. Herein, we report a 52-year-old male who presented with refractory gouty arthropathy for 30 years, manifested with a triad of chills, high fever and arthritis. He underwent several sessions of plasma adsorption, and his symptoms soon improved, along with a drop in inflammatory mediators. We conclude that plasma adsorption may be a promising and feasible treatment for refractory gout when conventional treatments are unsatisfactory or contraindicated.

Empagliflozin reduces kidney fibrosis and improves kidney function by alternative macrophage activation in rats with 5/6-nephrectomy.

BACKGROUND: Sodium glucose cotransporter 2 (SGLT2) inhibitors originally developed for the treatment of type 2 diabetes are clinically very effective drugs halting chronic kidney disease progression. The underlying mechanisms are, however, not fully understood. METHODS: We generated single-cell transcriptomes of kidneys from rats with 5/6 nephrectomy before and after SGLT2 inhibitors treatment by single-cell RNA sequencing. FINDINGS: Empagliflozin treatment decreased BUN, creatinine and urinary albumin excretion compared to placebo by 39.8%, 34.1%, and 55%, respectively (p < 0.01 in all cases). Renal interstitial fibrosis and glomerulosclerosis was likewise decreased by 51% and 66.8%; respectively (p < 0.05 in all cases). 14 distinct kidney cell clusters could be identified by scRNA-seq. The polarization of M2 macrophages from state 1 (CD206-CD68- M2 macrophages) to state 5 (CD206+CD68+ M2 macrophages) was the main pro-fibrotic process, as CD206+CD68+ M2 macrophages highly expressed fibrosis-promoting genes and can convert into fibrocytes. Empagliflozin remarkably inhibited the expression of fibrosis-promoting (IFG1 and TREM2) and polarization-associated genes (GPNMB, LGALS3, PRDX5, and CTSB) in CD206+CD68+ M2 macrophages and attenuated inflammatory signals from CD8+ effector T cells. The inhibitory effect of empagliflozin on CD206+CD68+ M2 macrophages polarization was mainly achieved by affecting mitophagy and mTOR pathways. INTERPRETATION: We propose that the beneficial effects of empagliflozin on kidney function and morphology in 5/6 nephrectomyiced rats with established CKD are at least partially due to an inhibition of CD206+CD68+ M2 macrophage polarization by targeting mTOR and mitophagy pathways and attenuating inflammatory signals from CD8+ effector T cells. FUNDINGS: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

[Prediction of mycophenolic acid exposure in renal transplantation recipients by artificial neural network].

The paper is aimed to establish an artificial neural network (ANN) for predicting mycophenolic acid (MPA) area under the plasma concentration-time curve (AUC) in renal transplantation recipients. 64 Chinese renal transplantation recipients receiving mycophenolate mofetil (MMF) were investigated. 10 timed samples were drawn at different days after transplantation. Plasma MPA concentration was determined by HPLC method and area under curve over the period of 0 to 12 h (AUC(0-12 h)) was calculated using the linear trapezoidal rule. ANN was established after network parameters were optimized using momentum method in combination with genetic algorithm. Furthermore, the predictive performance of ANN was compared with that of multiple linear regression (MLR). When using plasma MPA concentration of 0, 0.5, 2 h after MMF administration to predict MPA AUC(0-12 h), mean prediction error and mean absolute prediction error were -1.53% and 9.12%, respectively. Accuracy and precision of prediction by ANN were superior to that of MLR prediction, and similar results could be found when using plasma MPA concentration of 0, 0.5 h to predict MPA AUC(0-12h). The accuracy and precision of ANN prediction were superior to that of MLR prediction, and ANN can be used to predict MPA AUC(0-12 h).

MicroRNA-149 sensitizes colorectal cancer to radiotherapy by downregulating human epididymis protein 4.

Human epididymis protein 4 (HE4) was significantly up-regulated in colorectal cancer (CRC), while the potential relevance to radiation resistance of this phenomenon is still elusive. Relative expressions of target genes were quantified by real-time PCR. The protein level was determined by Western blot. The regulatory effect of miR-149 on WFDC2 (gene encoding HE4 protein) expression was analyzed by luciferase reporter assay. The response to radiation was evaluated by clonogenic assay in vitro and xenograft growth in vivo. WFDC2 was aberrantly up-regulated and miR-149 was down-regulated in CRC. MiR-149 repressed WFDC2 expression via directly targeting its 3'UTR region. The ectopic expression of miR-149 significantly sensitized CRC to radiation both in vitro and in vivo. Likewise, we further demonstrated that WFDC2-deficiency remarkably improved the radiation resistance in CRC. Simultaneously, WFDC2 rescue completely abolished the radiation sensitivity imposed by miR-149. Our data suggested that miR-149 sensitized CRC to radiation via directly inhibiting WFDC2/HE4, which would hold great promise for future therapeutic exploitations.

Molecular evolution and multilocus sequence typing of 145 strains of SARS-CoV.

In this study, we have identified 876 polymorphism sites in 145 complete or partial genomes of SARS-CoV available in the NCBI GenBank. One hundred and seventy-four of these sites existed in two or more SARS-CoV genome sequences. According to the sequence polymorphism, all SARS-CoVs can be divided into three groups: (I) group 1, animal-origin viruses (such as SARS-CoV SZ1, SZ3, SZ13 and SZ16); (II) group 2, all viruses with clinical origin during first epidemic; and (III) group 3, SARS-CoV GD03T0013. According to 10 special loci, group 2 again can be divided into genotypes C and T, which can be further divided into sub-genotypes C1-C4 and T1-T4. Positive Darwinian selections were identified between any pair of these three groups. Genotype C gives neutral selection. Genotype T, however, shows negative selection. By comparing the death rates of SARS patients in the different regions, it was found that the death rate caused by the viruses of the genotype C was lower than that of the genotype T. SARS-CoVs might originate from an unknown ancestor.

[The role of TGF-beta1/Smads in the development of peritoneal fibrosis induced by high glucose peritoneal dialysate and LPS].

OBJECTIVE: To investigate the expression and the potential role of TGF-beta/Smads in peritoneal fibrosis induced by high glucose dialysate and LPS in rats. METHODS: 24 male Sprague-Dawley rats were randomly allocated into four groups: control group, normal rats; LPS group: rats were treated with intraperitoneal injection of LPS (0.6 mg/kg body weight) on days 1, 3, 5, 7; dialysate Group: rats were treated with daily intraperitoneal injection of 4.25% peritoneal dialysate (100 ml/kg body weight) for 4 weeks; LPS + dialysate Group: daily intraperitoneal injection of 4.25% dialysate combined with four times injection of LPS (0.6 mg/kg body weight on days 1, 3, 5, 7) for 4 weeks. The parietal thickness was measured with masson stain. The expression of alpha-SMA, TGF-beta1, Smad 2/3, Smad 7 and ColI in peritoneal membrane was detected with confocal microscope by immuno-fluorescence, Western-blot and RT-PCR. RESULTS: Masson stain show the parietal thickness of the rats in all groups was significantly increased compared with control group and collagen deposition was evident in the thickened submesothelial compact zone. Parietal thickness of the rats in LPS + dialysate Group was most (vs LPS group: 41.5 +/- 3.3 microm vs 34.70 +/- 3.6 microm, P = 0.007, vs dialysate Group, 41.5 +/- 3.3 microm vs 20.2 +/- 3.6 microm, P = 0.000). The expression of alpha-SMA, Col I, TGF-beta1, Smad 3 was up-regulated in protein and mRNA level and the protein level of phosphorylated-Smad 2/3 was increased significantly. The most significant changes were found in LPS + dialysate Group. Compared with control group the mRNA and protein level of Smad7 was increased, but the protein ratio of phosphorylated Smad/Smad 7 in all groups was higher. Under electro-microscope, the mesothelial cells in LPS + dialysate Group had myofibroblast morphology with the presence of large bundles of actin microfilaments and dense bodies within the cytoplasm. CONCLUSIONS: High concentration glucose dialysate or LPS contributes to peritoneal fibrosis by stimulating TGF-beta/Smads signaling. 4.25% peritoneal dialysate can coordinate with LPS to activate TGF-beta/Smads signaling pathway and induce mesenchymal transdifferentiation and peritoneal fibrosis.

Lentiviral-mediated short hairpin RNA silencing of APE1 suppresses hepatocellular carcinoma proliferation and migration: A potential therapeutic target for hepatoma treatment.

Apurinic/apyrimidinic endonuclease-1 (APE1) is a protein involved in DNA repair and transcriptional regulation of gene expression. APE1 expression was reported to be correlated with poor prognosis in hepatocellular carcinoma (HCC) patients. Based on our previous study, we hypothesized that APE1 may be involved in the metastatic progression of HCC. Thus, the present study aimed to investigate the knockdown effect of APE1 using shRNA in HCC and demonstrate that silencing of APE1 in MHCC97-H cells can decrease the oncogenic transforming potential in vitro and reduce the growth of HCC tumor xenografts in vivo. Silencing of APE1 expression decreased the cell proliferation and survival, reduced the cell adhesion ability in Matrigel or fibronectin-coated plates and suppressed the cell migration and invasion in a Transwell assay of HCC cells. In the xenograft study, tumor growth was markedly inhibited in the APE1-silenced group. Silencing of APE1 in MHCC97-H cells decreased the oncogenic transforming potential in vitro and reduced the growth of HCC tumor xenografts in vivo. Inhibition of APE1 may present a novel therapeutic approach for the treatment of HCC.