Abnormal mRNA Splicing Effect of COL4A3 to COL4A5 Unclassified Variants.

Introduction: Genetic diagnosis of Alport syndrome (AS), which results from pathogenic variants in COL4A3, COL4A4, or COL4A5 genes, is hindered by large numbers of unclassified variants detected using next-generation sequencing (NGS). We examined the impact on splicing of variants of uncertain significance in COL4A3 to COL4A5. Methods: Nine unrelated patients with clinical diagnosis or suspicion of AS were enrolled according to the criteria. Their clinical and genetic data were collected. Blood and urine samples were obtained from the patients and their family members. Sanger sequencing was used to confirm the 9 COL4A3 to COL4A5 unclassified variants identified by NGS. COL4A3 to COL4A5 mRNAs from urine were analyzed using targeted reverse transcription polymerase chain reaction and direct sequencing. Results: Nine COL4A3 to COL4A5 unclassified variants were found to alter mRNAs splicing. Skipping of an exon or an exon fragment was induced by variants COL4A3 c.828+5G>A; COL4A4 c.3506-13_3528del; and COL4A5 c.451A>G (p. [Ile151Val]), c.2042-9 T>G, c.2689 G>C (p. [Glu897Gln]) and c.1033-10_1033-2delGGTAATAAA. Retention of an intron fragment was caused by variants COL4A3 c.3211-30G＞T, and COL4A5 c.4316-20T>A and c.1033-10 G>A, respectively. The 9 families in this study obtained genetic diagnosis of AS, including 3 with autosomal recessive AS and 6 with X-linked AS. Conclusions: Our findings demonstrate that urine mRNA analysis facilitates the identification of abnormal splicing of unclassified variants in Alport genes, which provides evidence of routine use of RNA analysis to improve genetic diagnosis of AS.

Intraindividual variations of urinary biomarkers in hospitalized children with glomerular diseases: a prospective observational study.

This study aimed to assess the intraindividual variations of urinary biomarkers in hospitalized children with glomerular diseases. Hospitalized children with glomerular diseases participated in the study. For each patient, an overnight (9:00 p.m.-7:00 a.m.) urine was collected, followed by a 24-h urine (classified into four distinct periods: morning 7:00 a.m.-12:00 p.m., afternoon 12:00 p.m.-4:00 p.m., evening 4:00 p.m.-9:00 p.m., and overnight 9:00 p.m.-7:00 a.m.). The concentrations of protein, albumin, N-acetyl-beta-D-glucosaminidase, and epidermal growth factor (EGF) were measured and normalized by three correction factors (creatinine, osmolality, or specific gravity, respectively). Additionally, the 2nd overnight urine sample was grouped into different aliquots according to centrifugation, additives, storage temperature, or delayed processing. Twenty (14 boys, 6 girls) children were enrolled, with an average age of 11.3 years. Among the three correction factors, creatinine-normalized biomarkers provided the best agreements among different periods over 24 h. There were significant diurnal variations during 24 h in the concentrations of urinary protein, albumin, N-acetyl-beta-D-glucosaminidase, and EGF (p = 0.001, p = 0.003, p = 0.003, and p = 0.003, respectively). Evening urine overestimated 24-h urinary protein and albumin, while overnight urine underestimated 24-h urinary albumin. Urinary EGF showed low variability within a day or between the 2 days (coefficients of variation 10.2% and 10.6%, respectively) and excellent agreements (intraclass correlation coefficients > 0.9) with 24-h urinary concentration. Furthermore, urinary EGF was not affected by centrifugation, additives, storage temperature, or delayed processing of urine samples (all p > 0.05).  Conclusion: Given the diurnal variations of urinary biomarkers, urine samples should be collected during the same time period in clinical practice if possible. The results also extend the evidence for urinary EGF as a relatively stable biomarker applied in the future clinical practice. What is Known: • Urinary biomarkers have been widely used or discussed in making diagnoses and therapy regimens and estimating the prognosis of pediatric glomerular diseases. It remains unclear whether their levels would be affected by the time of sample collection, processing methods, and storage conditions in hospitalized children with glomerular diseases. What is New: • The levels of both commonly used biomarkers and novel biomarkers exhibited diurnal variations in hospitalized children with glomerular diseases. • Our results extend the evidence for urinary EGF as a relatively stable biomarker applied in the future clinical practice.

Comparing recombinant human rabies monoclonal antibody (ormutivimab) with human rabies immunoglobulin (HRIG) for postexposure prophylaxis: A phase III, randomized, double-blind, non-inferiority trial.

OBJECTIVES: To evaluate the immunogenicity and safety of an anti-rabies monoclonal antibody (mAb), ormutivimab, compared with human rabies immunoglobulin (HRIG). METHODS: This phase III trial was designed as a randomized, double-blind, non-inferiority clinical trial in patients aged ≥18 years with suspected World Health Organization category Ⅲ rabies exposure. The participants were randomized 1:1 to ormutivimab and HRIG groups. After thorough wound washing and injection of ormutivimab/HRIG on day 0, the vaccination was administered on days 0, 3, 7, 14, and 28. The primary endpoint was the adjusted geometric mean concentration (GMC) of rabies virus-neutralizing activity (RVNA) on day 7. The endpoint of safety included the occurrence of adverse reactions and serious adverse events. RESULTS: A total of 720 participants were recruited. The adjusted-GMC of RVNA (0.41 IU/ml) on day 7 in ormutivimab group was not inferior to that in the HRIG group (0.41 IU/ml), with ratio of adjusted-GMC of 1.01 (95% confidence interval: 0.91, 1.14). The seroconversion rate of the ormutivimab group was higher than that of the HRIG group on days 7, 14, and 42. Most local injection sites and systemic adverse reactions reported from both groups were mild to moderate in severity. CONCLUSION: ormutivimab + vaccine can protect victims aged ≥18 years with category Ⅲ suspected rabies exposure as a component of postexposure prophylaxis. ormutivimab has a weaker influence on the immunity response of rabies vaccines. CLINICAL TRIALS REGISTRATION: ChiCTR1900021478 (the Chinese Clinical Trial Registry of World Health Organization).

Urinary epidermal growth factor predicts complete remission of proteinuria in Chinese children with IgA nephropathy.

BACKGROUND: This study investigated the association between urinary epidermal growth factor (EGF) and complete remission (CR) of proteinuria in children with IgA nephropathy (IgAN). METHODS: We included 108 patients from the Registry of IgA Nephropathy in Chinese Children. The urinary EGF at the baseline and follow-up were measured and normalized by urine creatinine (expressed as uEGF/Cr). The person-specific uEGF/Cr slopes were estimated using linear mixed-effects models for the subset of patients with longitudinal data of uEGF/Cr. Cox models were used to analyze the associations of baseline uEGF/Cr and uEGF/Cr slope with CR of proteinuria. RESULTS: Patients with high baseline uEGF/Cr were more likely to achieve CR of proteinuria (adjusted HR 2.24, 95% CI: 1.05-4.79). The addition of high baseline uEGF/Cr on the traditional parameters significantly improved the model fit for predicting CR of proteinuria. In the subset of patients with longitudinal data of uEGF/Cr, high uEGF/Cr slope was associated with a higher likelihood of CR of proteinuria (adjusted HR 4.03, 95% CI: 1.02-15.88). CONCLUSIONS: Urinary EGF may be a useful noninvasive biomarker for predicting and monitoring CR of proteinuria in children with IgAN. IMPACT: High levels of baseline uEGF/Cr (>21.45 ng/mg) could serve as an independent predictor for CR of proteinuria. The addition of baseline uEGF/Cr on the traditional clinical pathological parameters significantly improved the fitting ability for the prediction of CR of proteinuria. Longitudinal data of uEGF/Cr were also independently associated with CR of proteinuria. Our study provides evidence that urinary EGF may be a useful noninvasive biomarker in the prediction of CR of proteinuria as well as monitoring therapeutic response, thus guiding treatment strategies in clinical practice for children with IgAN.

A novel prognostic and therapeutic target biomarker based on complement-related gene signature in gastric cancer.

Background: Gastric cancer (GC) is one of the most prevalent cancer types that reduce human life expectancy. The current tumor-node-metastasis (TNM) staging system is inadequate in identifying higher or lower risk of GC patients because of tumor heterogeneity. Research shows that complement plays a dual role in the tumor development and progression of GC. Methods: We downloaded GC data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). A complement-related risk signature was constructed through bioinformatics analysis. Subsequently, the predictive ability of this signature was validated with GSE84437 dataset, and a nomogram integrating risk score and common clinical factors was established. Besides, we evaluated the association of risk score with the immune and stromal cell infiltration in TCGA. Furthermore, immunotherapy response prediction and drug susceptibility analysis were conducted to access the ability of the risk signature in predicting the therapeutic effect. Results: A complement-related gene (CRG) signature, based on six genes (SPLG, C9, ITIH1, ZFPM2, CD36, and SERPINE1), was established. In both the training and validation sets, the overall survival of GC patients in the high-risk group was lower than that of the low-risk group, and the nomogram to predict the 1-, 2-, and 3-year survival rates of GC patients was developed. In addition, CIBERSORT algorithm showed the high-risk patients had higher levels of immune cell infiltration than low-risk patients, and the ESTIMATE results implied that the high-risk group had more stromal component in tumor microenvironment. Besides, compared to the low-risk group, there were higher expressions of most immune checkpoint genes and HLA genes in the high-risk group, and the high-risk patients showed higher sensitivity to the chemotherapy and targeted drugs (axitinib, dasatinib, pazopanib, saracatinib, sunitinib and temsirolimus). Conclusions: The novel CRG signature may act as a reliable, efficient tool for prognostic prediction and treatment guidance in future clinical practice.

Comparison of diagnostic accuracy between linear EUS and miniprobe EUS for submucosal invasion in suspected cases of early gastric cancer

Objective: this study assessed the accuracy of linear endoscopic ultrasound (EUS) to diagnose submucosal (SM)invasion and compared linear EUS with miniprobe EUS insuspected early gastric cancer (EGC) patients.Methods: patients diagnosed with biopsy-verified suspected EGC were analyzed retrospectively. All cases wereexamined by linear EUS or miniprobe EUS for preoperativediagnosis of invasion depth and underwent endoscopic orsurgical treatment for radical resection. The invasion depthevaluated by EUS and pathology were categorized as noinvasion of SM and invasion of SM or deeper. The diagnosis of EUS was compared with postoperative pathologyresults.Results: a total of 105 patients were included in the finalanalysis. The overall prediction accuracy of linear EUS(n = 57) for SM invasion in suspected EGC was higherthan that of miniprobe EUS (n = 48), although no statistically significant differences were noted (82.5 % vs 72.9 %,p = 0.344). The negative predictive value (NPV) of linear EUSwas significantly higher than that of miniprobe EUS (100 %vs 82.8 %, p = 0.037). The binary logistic regression analysis showed that tumor size (p = 0.036), the presence of ulceration (p < 0.001) and EUS type (p = 0.027) were independentrisk factors for the diagnosis of SM invasion by EUS. Thearea under the receiver operating curve (ROC) was 0.889and 0.719 for linear and miniprobe EUS, respectively.Conclusion: linear EUS diagnosed suspected EGC for SMinvasion with a higher accuracy than miniprobe EUS. Inaddition, large and ulcerative lesions may lead to overestimation. (AU)

Comparison of diagnostic accuracy between linear EUS and miniprobe EUS for submucosal invasion in suspected cases of early gastric cancer.

OBJECTIVE: This study assessed the accuracy of linear endoscopic ultrasound (EUS) to diagnose submucosal (SM) invasion and compared linear EUS with mini-probe EUS in suspected early gastric cancer (EGC) patients. METHODS: Patients diagnosed with biopsy-verified suspected EGC were analyzed retrospectively. All cases were examined by linear EUS or miniprobe EUS for preoperative diagnosis of invasion depth and underwent endoscopic or surgical treatment for radical resection. The invasion depth evaluated by EUS and pathology were categorized as no invasion of SM and invasion of SM or deeper. The diagnosis of EUS was compared with postoperative pathology results. RESULTS: A total of 105 patients were included in the final analysis. The overall prediction accuracy of linear EUS (n = 57) for SM invasion in suspected EGC was higher than that of mini-probe EUS (n = 48), although no statistically significant differences were noted (82.5% vs 72.9%, p = 0.344). The negative predictive value (NPV) of linear EUS was significantly higher than that of mini-probe EUS (100% vs 82.8%, p = 0.037). The binary logistic regression analysis showed that tumor size (p = 0.036), the presence of ulceration (p < 0.001) and EUS type (p = 0.027) were independent risk factors for the diagnosis of SM invasion by EUS. The area under the receiver operating curve (ROC) was 0.889 and 0.719 for linear and mini-probe EUS, respectively. CONCLUSION: Linear EUS diagnosed suspected EGC for SM invasion with a higher accuracy than mini-probe EUS. In addition, large and ulcerative lesions may lead to overestimation.

Increased METTL3-mediated m6A methylation inhibits embryo implantation by repressing HOXA10 expression in recurrent implantation failure.

BACKGROUND: Recurrent implantation failure (RIF) is a major limitation of assisted reproductive technology, which is associated with impaired endometrial receptivity. Although N6-methyladenosine (m6A) has been demonstrated to be involved in various biological processes, its potential role in the endometrium of women with RIF has been poorly studied. METHODS: Global m6A levels and major m6A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m6A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m6A modification on embryo attachment were evaluated by an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, and the expression levels of homeo box A10 (HOXA10, a well-characterized marker of endometrial receptivity) and its downstream targets were evaluated by quantitative real-time PCR and Western blotting in METTL3-overexpressing Ishikawa cells. The molecular mechanism for METTL3 regulating HOXA10 expression was determined by methylated RNA immunoprecipitation assay and transcription inhibition assay. RESULTS: Global m6A methylation and METTL3 expression were significantly increased in the endometrial tissues from women with RIF compared with the controls. Overexpression of METTL3 in Ishikawa cells significantly decreased the ration of BeWo spheroid attachment, and inhibited HOXA10 expression with downstream decreased ß3-integrin and increased empty spiracles homeobox 2 expression. METTL3 catalyzed the m6A methylation of HOXA10 mRNA and contributed to its decay with shortened half-life. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro. CONCLUSION: Increased METTL3-mediated m6A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.

C-X-C Motif Chemokine Ligand 16 Is a Potent Predictor of Outcomes in Dialysis Patients.

INTRODUCTION: C-X-C motif chemokine ligand 16 (CXCL16) is an inflammatory marker that has been found to be predictive of outcomes in patients with cardiovascular disease. Our previous work has also demonstrated its relation to cardiac injury in dialysis patients. However, it is yet unclear whether there is an association between CXCL16 and adverse outcomes in dialysis patients. We aimed to evaluate its prognostic value along with several traditional inflammatory markers in the current study. METHODS: This is a multicenter longitudinal study of prevalent dialysis patients. Circulating inflammatory markers including CXCL16, C-reactive protein (CRP), tumor necrosis factor-α, and interleukin-6 (IL-6) were measured using a multiplex assay. The primary outcomes were all-cause mortality and a composite of major adverse cardiovascular events (MACEs). The associations between biomarkers and outcomes were analyzed using Cox proportional hazards regression models. RESULTS: Of the 366 participants with available plasma samples, the average age was 52.5 (±12.1) years, and there were 160 (43.7%) female participants. For all-cause mortality, logarithmically transformed CXCL16, IL-6, and CRP were independent predictors after adjustment for covariates. When the 3 markers were included in the same model, CXCL16 was the only one remaining its significance. For MACEs, logarithmically transformed CXCL16 and IL-6 were significant predictors when analyzed separately and CXCL16 was an independent predictor even after adjustment for IL-6. When the biomarkers were analyzed as categorical variables, only CXCL16 was associated with both outcomes. Adding CXCL16 to established risk factors improved risk prediction as revealed by Net Reclassification Index (NRI). CONCLUSION: Using a multimarker approach, we determined that CXCL16 is a potent predictor of all-cause mortality and cardiovascular events in dialysis patients. Our data suggest CXCL16 may improve risk stratification and could be a potential interventional target.

Immunogenicity and safety of the inactivated poliomyelitis vaccine made from Sabin strains in a phase IV clinical trial for the vaccination of a large population.

As a recently launched novel vaccine used as one of the vaccines for the final eradication of polios worldwide, complete data on the consistency and immunogenicity characteristics of the inactivated poliomyelitis vaccine made from the Sabin strain (sIPV) and its safety in large-scale populations are required to support the future use of this vaccine worldwide. A phase IV clinical trial was conducted to perform an immunogenicity evaluation of lot-to-lot consistency of three commercial batches of sIPV in 1200 infants and to investigate the vaccine's safety on a large-scale in 20,019 infants for active monitoring and 29,683 infants for passive monitoring through the Adverse Event Following Immunization (AEFI) reporting system in China. In the immunogenicity evaluation, the average seroconversion rates for type I, type II and type III of the three groups were 99.83%, 98.93% and 99.44%, respectively. No differences in the seroconversion rate and the GMT ratios were noted in the pair-to-pair comparisons. In the large-scale safety evaluation, most adverse reactions occurred 0-30 days after the first doses, and the common local and systemic reactions were similar to those in the phase III clinical trial, with low incidence in both activated and passive monitoring. In conclusion, sIPV exhibits good lot-to-lot consistency and safety in large-scale populations; thus, it is qualified to serve as one of the vaccines for use in eradicating all wild and vaccine-derived polioviruses worldwide in the near future. Clinic Trial Registration. NCT04224519 and NCT04220515.

The general public new views on deceased organ donation in China.

BACKGROUND: The organ donation in China has developed rapidly since fully launched donations after citizens death in 2015. This study was conducted to evaluate how the Chinese general public views changed on deceased organ donation, and to improve the donation process. METHODS: A total of 110 eligible studies, including 103, 410 individuals, were selected to analyze through searching PubMed, CBMdisc, CQVIP, CNKI, and Wanfang Data from Jan 1, 1990 to May 31, 2019. The pooled proportions (and 95% CIs) of cognition, attitudes and willingness related to organ donation were calculated using the Freeman-Tukey double arcsine transformation. RESULTS: The pooled proportions of knowing about organ donation and willing to donate increased from 84.6% (73.0-93.4) and 32.4% (23.9-41.6) before 2015, to 86.4% (74.5-95.1) and 39.9% (32.8-47.2) after 2015, respectively. The willingness to posthumous organ donation for cornea, heart, kidney, and liver had a significant improvement. Especially, the proportion of willingness to donate cornea increased to 56.0% (43.3-68.3) after 2015, from 39.2% (31.2-47.4) before 2015. However, although 69.7% (62.7-76.4) of participants approved the deceased organ donation, only 35.6% (29.7-41.8) and 43.9% (37.2-50.8) were willing to donate their own and relatives organs postmortem, respectively. The leading reasons for refraining from donating organs postmortem were distrusting the medical professionals (49.8%, 35.2-64.4) and traditional Chinese values (40.6%, 32.4-49.0). Popularizing knowledge about organ donation (61.5%, 45.7-76.1), humanitarian aid (57.1%, 48.8-65.3), and priority of using donated organs for relatives (53.1%, 30.8-74.7) were the applauded strategies to improve the willingness to posthumous organ donation. CONCLUSIONS: The willingness toward posthumous organ donation has a significant improvement among Chinese general public since 2015, however, several important measures still need to be taken to promote the favorable attitudes and willingness toward organ donation.

Adverse drug reactions and nursing interventions in the treatment of non-Hodgkin's lymphoma by Aidi injection.

BACKGROUND: To explore adverse drug reactions (ADRs) and the effects of nursing interventions after Aidi injection for the treatment of non-Hodgkin's lymphoma (NHL). METHODS: A total of 104 NHL patients treated in our hospital from March 2019 to March 2020 were selected. All patients underwent conventional chemotherapy, with a concomitant Aidi injection administered at different doses (40, 60, 80, and 100 mL). ADRs were analyzed for all patients, and the clinical outcomes of ADRs were recorded after specific nursing interventions were performed. RESULTS: A total of 17 NHL patients had ADRs, with a total incidence rate of 15.89% (total of 30 ADRs). In terms of different types of ADRs, inappetence accounted for the largest proportion, followed by skin pruritus and fever, phlebitis, nausea and vomiting, and chest distress and/or palpitation, the last of which shared the same proportion as chills and/or low fever and urticaria. The incidence rate of ADRs (total of 18 ADRs, 60.00%) was higher in NHL patients who were given 100 mL of Aidi injection. In terms of age and gender distribution of ADRs, there were 10 males (55.56%) and 7 females (44.44%), and ADRs were more common in patients aged 46-60 years old (total of 6 ADRs, 35.29%). Aidi injection mainly induced mild ADRs (total of 22 ADRs, 73.33%), and the resulting ADRs mostly occurred for <2 hours (total of 19 ADRs, 60.00%). After the specific nursing interventions were performed, no deaths due to ADRs occurred, and 12 (64.71%) cases were cured, 5 (29.41%) cases improved, and 1 (5.88%) case had no progression. CONCLUSIONS: Particular attention should be paid to ADRs in the treatment of NHL patients with Aidi injection. After ADRs occur, specific nursing interventions can aid in recovery and lead to improvements in prognosis.

[Efficacy and safety of steroid combined with immunosuppressants in the treatment of primary IgA nephropathy in children: a Meta analysis].

OBJECTIVE: To systematically evaluate the efficacy and safety of steroid combined with immunosuppressants in the treatment of primary IgA nephropathy in children. METHODS: English and Chinese electronic databases were searched to include the studies on the efficacy and safety of steroid combined with immunosuppressants versus steroid alone in the treatment of primary IgA nephropathy in children. Outcome measures included proteinuria remission rate, urinary protein quantification, incidence of adverse events, estimated glomerular filtration rate, and incidence of renal dysfunction. Review Manager 5.3 software was used for data analysis. RESULTS: A total of 7 studies with 381 children were included. The children had moderate to severe proteinuria. The Meta analysis showed that compared with the steroid alone group, the steroid combined with immunosuppressants group achieved a significantly higher rate of proteinuria remission (RR=1.36, 95%CI: 1.19-1.55, P<0.001) and significantly lower urinary protein quantification (SMD=-0.82, 95%CI: -1.23 to -0.41, P<0.001). There was no significant difference in the incidence rate of adverse events between the two groups (RR=1.28, 95%CI: 0.92-1.77, P=0.14). CONCLUSIONS: The current evidence shows that for children with primary IgA nephropathy who have moderate to severe proteinuria, steroid combined with immunosuppressants has a better effect than steroid alone and does not increase the incidence rate of adverse events.

Apigenin suppresses the apoptosis of H9C2 rat cardiomyocytes subjected to myocardial ischemia­reperfusion injury via upregulation of the PI3K/Akt pathway.

Apigenin, a flavonoid with multiple physiological and pharmacological activities, is associated with the prevention of cardiovascular diseases. The present study aimed to examine the roles and mechanisms of apigenin in the apoptosis of H9C2 rat cardiomyocytes, which were subjected to myocardial ischemia­reperfusion (MI/R) injury. Cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and cellular apoptosis were evaluated using cell counting kit­8 assays and flow cytometry. The content/activity of oxidative stress markers was determined using commercial kits. Western blot analysis and reverse transcription­quantitative polymerase chain reaction assays were used to measure protein and mRNA expression, respectively. The results demonstrated that apigenin had limited cytotoxicity on the viability of H9C2 rat cardiomyocytes. Apigenin reduced the oxidative stress, ROS production and cellular apoptotic capacity of MI/R­induced H9C2 cells. Apigenin additionally increased the MMP level of MI/R­induced H9C2 cells. Furthermore, apigenin modulated apoptosis­associated protein expression and phosphatidylinositol 3'-kinase (PI3K)/RAC­α serine/threonine­protein kinase (Akt) signaling in MI/R­induced H9C2 cells. Treatment with LY294002 reversed the anti­apoptotic effect of apigenin. In conclusion, apigenin suppressed the apoptosis of H9C2 cells that were subjected to MI/R injury by activating the PI3K/Akt pathway. It was suggested that apigenin may be effective as an MI/R therapy.

Emodin, A Chinese Herbal Medicine, Inhibits Reoxygenation-Induced Injury in Cultured Human Aortic Endothelial Cells by Regulating the Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathway.

BACKGROUND Ischemia-reperfusion injury is associated with vascular dysfunction. The aim of this study was to investigate the role of emodin, a Chinese herbal medicine, in hypoxia-reoxygenation injury in cultured human aortic endothelial cells (HAECs) and its effects on the expression of the peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS) signaling pathway. MATERIAL AND METHODS An in vitro hypoxia-reoxygenation model used cultured human aortic endothelial cells (HAECs). A colorimetric method evaluated the activity of peroxisome proliferator-activated receptor-γ (PPAR-γ). Phosphorylation of PPAR-γ and endothelial nitric oxide synthase (eNOS) were measured by Western blotting. Expression of inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Nitric oxide (NO) production was detected by diaminofluorescein-FM diacetate (DAF-FM DA) fluorescence. Immunoprecipitation was used to evaluate the molecular coupling of heat shock protein (HSP)90 and eNOS. RESULTS Hypoxia-reoxygenation injury of HAECs reduced the activity and phosphorylation of PPAR-γ, and eNOS, NO production, and HSP90/eNOS molecular coupling in a time-dependent manner. Hypoxia-reoxygenation increased the levels of inflammatory cytokines TNF-α, IL-6, and IL-8 in a time-dependent manner. Emodin treatment recovered PPAR-γ activity and phosphorylation, eNOS phosphorylation, and HSP90/eNOS coupling in HAECS in a concentration-dependent manner, which was reversed by the PPAR-γ inhibitor GW9662, and the eNOS inhibitor, L-NAME. The recovery of HSP90/eNOS coupling by emodin was impaired by GW9662 treatment. CONCLUSIONS An in vitro hypoxia-reoxygenation (ischemia-reperfusion injury) model of induction of endothelial cell inflammatory mediators showed that emodin recovered the PPAR-γ and eNOS pathway activity.

SIRT6 inhibits growth of gastric cancer by inhibiting JAK2/STAT3 pathway.

SIRT6, a member of NAD positivity-dependent class III deacetylase sirtuin family, played versatile roles in human cancers. However, the expression and biological function of SIRT6 in gastric cancer (GC) remain to be investigated. In this study, we found that SIRT6 expression level was decreased in gastric cancer tissues and cell lines. Decreased SIRT6 expression was associated with unfavorable clinical parameters including tumor differentiation, tumor size and TNM stage. Importantly, decreased level of SIRT6 was associated with decreased rate of overall survival (OS) and disease­free survival (DFS). Functionally, overexpression of SIRT6 in both BGC823 and SGC7901 cells inhibited cell viability and proliferation of GC cells. Furthermore, overexpression of SIRT6 in both BGC823 and SGC7901 cells prevented the cell cycle progress and increased cell apoptosis. In vivo experiments demonstrated that forced expression of SIRT6 in SGC-7901 cells inhibited the growth of SGC7901 cells in nude mice. Furthermore, the IHC staining for GC tissues showed that expression level of SIRT6 was decreased in GC tissues while the expression level of p-STAT3 was increased in GC tissues. GC tissues with high SIRT6 level showed significantly decreased level of p-STAT3. Mechanically, we demonstrated that SIRT6 blocked the activation of JAK2/STAT3 and inhibited the expression of cyclin D1 and Bcl2 which were downstream targets of JAK2/STAT3 pathway. Taken together, this study indicates that SIRT6 inhibits the growth of gastric cancer by inhibiting JAK2/STAT3 pathway.

Interaction of Soluble CXC Ligand 16 and Cardiac Injury Markers in Hemodialysis Patients.

BACKGROUND: Recent data suggest that there is a pathogenic role for CXC ligand 16 (CXCL16) in cardiovascular diseases. Little is known about circulating CXCL16 in patients with kidney dysfunction. We explored the relationships of plasma CXCL16 with cardiac injury markers in a group of dialysis patients. METHODS: Plasma CXCL16 and C-reactive protein (CRP) were measured in 366 patients who were on maintenance hemodialysis. Cardiac injury was evaluated via measurements of the circulating B-type natriuretic peptide (BNP), N-terminal prohormone of brain natriuretic peptide (NT proBNP), Troponin I (TnI), and Troponin T (TnT). Sixty healthy subjects who were frequency matched with the patients on the basis of age and gender were recruited as healthy controls. RESULTS: The mean age of the patients was 52.5 ± 12.1 years and 56.3% were male. Circulating CXCL16 was significantly higher in the patients than in the controls (patients vs. CONTROLS: 477.3 (367.0-647.1) pg/mL vs. 229.5 (203.8-254.5) pg/mL; p < 0.001). The log-transformed (log-) CXCL16 level was correlated with all 4 cardiac markers (log-BNP, log-NTproBNP, log-TnI, and log-TnT) with high levels of significance (all p < 0.001), even after extensive controls for the covariates. In contrast, CRP was correlated only with BNP (marginally) and NT proBNP and was not correlated with troponins. CONCLUSION: We showed, for the first time, highly significant relationships of circulating CXCL16 level with cardiac injury markers in dialysis patients. Our data suggest that circulating CXCL16 is possibly involved in the pathological process of cardiovascular damage in dialysis patients and may serve as a therapeutic target for cardiac protection in these patients.

Association of adipokines with blood pressure, arterial elasticity and cardiac markers in dialysis patients: cross-sectional analysis of baseline data from a cohort study.

BACKGROUND: Adipokines are a set of cytokines secreted by white adipose tissue that have been suggested to be involved in the development of cardiovascular diseases. We aimed to evaluate the cross-sectional associations of a panel of representative adipokines with cardiovascular measures in a cohort of hemodialysis patients. METHODS: We measured plasma adiponectin, resistin, plasminogen activator inhibitor-1 (PAI-1), leptin, monocyte chemotactic protein 1 (MCP-1) and adipsin levels in 366 dialysis patients and 60 healthy controls. The associations of these adipokines with systolic blood pressure (assessed by ambulatory blood pressure monitoring), pulse wave velocity (PWV) and cardiac markers (BNP, NT-proBNP, Troponin I, Troponin T) in these patients were determined by general linear models with stepwise adjustment for covariates. RESULTS: In unadjusted comparison with controls, dialysis patients showed increased adiponectin, resistin, MCP-1 and adipsin levels, decreased PAI-1 concentrations (all p <0.001) and similar leptin levels (p = 0.82). On adjustment for body mass index and diabetes, however, the PAI-1 level was comparable between group (p = 0.06), whereas leptin levels became significantly higher in the patients(p <0.001). Higher adiponectin, lower PAI-1 and leptin levels were associated with higher systolic blood pressure, even after extensive adjustment (all p ≤ 0.01). Adiponectin was also consistently and inversely associated with PWV in fully adjusted models (p = 0.003). Resistin, PAI-1, leptin and adipsin showed negative associations with one or more circulating cardiac markers (all p ≤ 0.02). CONCLUSIONS: We found significant associations between adipokines and cardiovascular measures. Our data suggest the possible involvement of adipokines in cardiovascular modulation in dialysis patients.

Ambulatory arterial stiffness index and its role in assessing arterial stiffness in dialysis patients.

OBJECTIVE: Ambulatory arterial stiffness index (AASI) is a parameter derived from ambulatory blood pressure (ABP) readings. It is calculated as 1 minus the linear slope of DBP on SBP. We tested its value in assessing arterial stiffness in dialysis patients. METHODS: We performed a cross-sectional analysis of the baseline data from a cohort study. A total of 344 patients on maintenance hemodialysis from six tertiary hospitals were included. All patients underwent ABP monitoring and carotid-femoral pulse wave velocity (cfPWV) measurement. Clinical determinants of AASI were analyzed, and the ability of AASI for assessing arterial stiffness was compared with ambulatory pulse pressure (PP). RESULTS: Multiple regression analysis revealed that ambulatory PP (ß = 0.003), current smoker (ß = -0.069), age (ß = 0.003) and ambulatory SBP (ß = 0.001) were independent determinants of AASI. Ambulatory PP correlates better with cfPWV than AASI (r = 0.28 for AASI and 0.59 for PP; P for difference: <0.001). When cfPWV was treated as a categorical variable, receiver operating characteristic curve analysis also showed a more potent predictive value of PP over AASI (area under the curve: 0.64 for AASI, 0.80 for PP; P for difference: <0.001). Net reclassification improvement and integrated discrimination improvement analysis demonstrated no added predictive value of AASI to PP (net reclassification improvement = -2.2%, P = 0.26; integrated discrimination improvement = 0.001, P = 0.51). Sensitivity analysis in patients with more ABP readings (≥49) yielded similar results. CONCLUSION: For dialysis patients, AASI has very limited value in assessing arterial stiffness, whether used alone or added to PP. Our results suggest that this index should not be used as a surrogate marker of arterial stiffness for dialysis patients in future practice and studies.

Involvement of endoplasmic reticulum stress in formalin-induced pain is attenuated by 4-phenylbutyric acid.

BACKGROUND: Endoplasmic reticulum (ER) stress is involved in many neurological and inflammatory responses. Peripheral inflammatory responses can induce central sensitization and trigger inflammatory pain. However, there is little research on the relationship between ER stress and inflammatory pain. In this study, we examined whether the ER stress response is involved in peripheral inflammatory pain using a formalin-induced rat pain model. METHODS: Rats were divided into the following five groups: control, formalin, formalin + vehicle, formalin + 4-phenylbutyric acid (4-PBA) (40 mg/kg) and formalin + 4-PBA (100 mg/kg). Formalin-induced pain was assessed behaviorally by recording licking activity. The expression levels of immunoglobulin-binding protein (BIP), activating transcription factor-6 (ATF6), phosphorylated inositol-requiring enzyme-1 (p-IRE1), phosphorylated protein kinase RNA-like ER kinase (p-PERK) and c-fos were quantitatively assessed by Western blot, and the distribution of BIP, ATF6 and c-fos in the lumbar enlargement of spinal cord were identified by immunohistochemistry in spinal dorsal horn slices. In addition, the concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) in the spinal cord were tested by biochemical measurement and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Intraperitoneal injection of 4-PBA at the dose of 100 mg/kg before formalin injection significantly decreased nociceptive behavior in the second phase compared with control, formalin, formalin + vehicle and formalin + 4-PBA (40 mg/kg) (P<0.05). Western blot showed that formalin injection significantly upregulated the expression of BIP, ATF6, p-PERK and c-fos in the spinal cord. This upregulation was reduced by peritoneal injection of 4-PBA (P<0.05), while expression of p-IRE1 was not altered by formalin treatment. Immunohistochemistry revealed markedly increased staining density for BIP, ATF6 and c-fos in the superficial spinal dorsal horn after formalin injection. This was significantly decreased by administration of 4-PBA (P<0.05). Compared with the formalin + vehicle group, 4-PBA inhibited the release of NO and PGE2 in the spinal cord (P<0.05). CONCLUSION: These results suggest that ER stress is involved in formalin-induced inflammatory pain and that inhibition of ER stress may attenuate central sensitization induced by peripheral inflammatory stimulation.