Development and evaluation of an external quality control and internal quality control containing real-time RT-PCR assay for the detection of o'nyong-nyong virus.

O'nyong-nyong fever is a mosquito-borne tropical viral disease while few molecular diagnostic tools have been established for its surveillance until now. In the current study, a single-step, dual-color real-time reverse transcription polymerase chain reaction (RT-PCR) assay which contained both external quality control (EQC) and internal quality control (IQC) prepared by armored RNA technique was developed and evaluated for the detection of o'nyong-nyong virus (ONNV). Results showed that the assay was established successfully without cross-reaction with genetically related or symptom-alike diseases, which showed high specificity of the assay. The coefficient of variation of the assay was 0.97%, far less than 5%, indicating good repeatability of the assay. The lower limit of detection of the assay could reach as low as 100 copies of genome equivalent. During evaluation, the assay could correctly detect ONNV from spiked human serum samples and Anopheles species mosquito samples, while no ONNV positive was observed either from serum samples of patients with acute febrile illness or from local Anopheles species mosquitoes, suggesting no ONNV had been transmitted locally. In conclusion, the assay could potentially provide a valuable platform for ONNV molecular detection, which may improve the preparedness for future o'nyong-nyong fever outbreaks.

Impact of minimally invasive total mesoesophageal excision and minimally invasive esophagectomy on failure patterns of locally advanced esophageal squamous cell carcinoma: a matched cohort study with long-term follow-up.

BACKGROUND: The effects of minimally invasive total mesoesophageal excision (MITME) on the long-term prognosis of locally advanced esophageal squamous cell carcinoma (ESCC) remain unknown. The objective of this study was to compare the static and dynamic failure patterns of MITME and minimally invasive esophagectomy (MIE) for locally advanced ESCC. METHODS: We use propensity score matching (PSM) method to analyze the postoperative failure patterns of the two groups. Cumulative event curves were analyzed for cumulative incidence of failure between different groups, and independent prognostic factors were assessed using time-dependent multivariate analyses. The risk of dynamic failure calculated at 12-month intervals was compared between the two groups using the lifetime table. RESULTS: A total of 366 ESCC patients were studied by 1:1 PSM for T stage and TNM stage (MITME group, n = 183; MIE group, n = 183). In the matched cohort, there was significant differences between the MITME and MIE groups in the failure pattern of regional lymph node recurrence (0.5 vs 3.8%, P = 0.032) and non-tumor death (10.9 vs 31.7%, P < 0.001). The cumulative event curve found that the 5-year cumulative failure rate was lower in the MITME group than in the MIE group (3.3 vs 17.1%, P = 0.026) after 5 years of survival. In addition, multivariate Cox regression analysis showed that MIE was an independent poor prognostic factor for a high cumulative failure rate in locally advanced ESCC patients at 5 years after surgery (HR:4.110; 95% CI 1.047-16.135; P = 0.043). The dynamic risk curve showed that the MITME group had a lower risk of failure within 5 years after surgery than the MIE group. CONCLUSION: Considering that MITME can significantly improve the postoperative failure pattern and the benefit lasts for at least 5 years, it is feasible to use MITME as a treatment for locally advanced ESCC.

Cellular phenotypic transitions in diabetic nephropathy: An update.

Diabetic nephropathy (DN) is a major cause of morbidity and mortality in diabetes and is the most common cause of end stage renal disease (ESRD). Renal fibrosis is the final pathological change in DN. It is widely believed that cellular phenotypic switching is the cause of renal fibrosis in diabetic nephropathy. Several types of kidney cells undergo activation and differentiation and become reprogrammed to express markers of mesenchymal cells or podocyte-like cells. However, the development of targeted therapy for DN has not yet been identified. Here, we discussed the pathophysiologic changes of DN and delineated the possible origins that contribute to myofibroblasts and podocytes through phenotypic transitions. We also highlight the molecular signaling pathways involved in the phenotypic transition, which would provide valuable information for the activation of phenotypic switching and designing effective therapies for DN.

Flavonoids in Treatment of Chronic Kidney Disease.

Chronic kidney disease (CKD) is a progressive systemic disease, which changes the function and structure of the kidneys irreversibly over months or years. The final common pathological manifestation of chronic kidney disease is renal fibrosis and is characterized by glomerulosclerosis, tubular atrophy, and interstitial fibrosis. In recent years, numerous studies have reported the therapeutic benefits of natural products against modern diseases. Substantial attention has been focused on the biological role of polyphenols, in particular flavonoids, presenting broadly in plants and diets, referring to thousands of plant compounds with a common basic structure. Evidence-based pharmacological data have shown that flavonoids play an important role in preventing and managing CKD and renal fibrosis. These compounds can prevent renal dysfunction and improve renal function by blocking or suppressing deleterious pathways such as oxidative stress and inflammation. In this review, we summarize the function and beneficial properties of common flavonoids for the treatment of CKD and the relative risk factors of CKD.

Textbook outcome after minimally invasive esophagectomy is an important prognostic indicator for predicting long-term oncological outcomes with locally advanced esophageal squamous cell carcinoma.

Background: The textbook outcome (TO) emerges as a novel prognostic factor in surgical oncology. The present study aimed to evaluate the effect of TO on the risk of death and recurrence in patients with esophageal squamous cell carcinoma (ESCC) after minimally invasive esophagectomy (MIE). Methods: The study involved retrospective analysis of 528 patients with ESCC who were subjected to MIE from January 2011 to December 2017. TO included 8 parameters: complete resection; microscopically tumor-negative resection margins (R0); ≥15 lymph nodes removed and examined; no serious postoperative complications; no postoperative intervention; no re-admission to the intensive care unit (ICU); hospital stay ≤21 days; and no readmission ≤30 days. The Cox and logistic regression model were used to analyze the prognostic factors of survival and risk factors for TO. Results: Among the 528 patients with ESCC who were subjected to MIE, 53.2% reached TO. In the case of patients with locally advanced ESCC, 5-year overall survival (OS) was 51.1% (41.2-61.2%) for the TO group but 33.7% (23.7-43.7%) for the non-TO group (HR =0.644, 95% CI: 0.449-0.924, P=0.015). Similarly, 5-year disease-free survival (DFS) was 47.6% (38.0-57.2%) for the TO group but 29.1% (20.1-38.1%) for the non-TO group (HR =0.671, 95% CI: 0.479-0.940, P=0.018). In addition, 5-year recurrence-free survival (RFS) was 62.9% (53.7-72.1%) for the TO group but 39.8% (29.4-50.2%) for the non-TO group (HR =0.606, 95% CI: 0.407-0.902, P=0.012). Multivariate logistic regression analysis further showed that age, American Society of Anesthesiology (ASA) score, intraoperative blood loss, and smoking status acted as independent risk factors for TO. The results of the multivariate analysis assisted in the establishment of a nomogram for the prediction of TO occurrence. This nomogram exhibited satisfactory consistency and prediction ability [area under the receiving operator characteristic (AUROC) =0.717]. Conclusions: The present study showed that achieving of TO after MIE improves survival rate and reduce the recurrence rate in patients with locally advanced ESCC. The study further determined the independent factors associated with TO achievement and established a prediction model.

Mitochondrial Oxidative Stress and Cell Death in Podocytopathies.

Podocytopathies are kidney diseases that are driven by podocyte injury with proteinuria and proteinuria-related symptoms as the main clinical presentations. Albeit podocytopathies are the major contributors to end-stage kidney disease, the underlying molecular mechanisms of podocyte injury remain to be elucidated. Mitochondrial oxidative stress is associated with kidney diseases, and increasing evidence suggests that oxidative stress plays a vital role in the pathogenesis of podocytopathies. Accumulating evidence has placed mitochondrial oxidative stress in the focus of cell death research. Excessive generated reactive oxygen species over antioxidant defense under pathological conditions lead to oxidative damage to cellular components and regulate cell death in the podocyte. Conversely, exogenous antioxidants can protect podocyte from cell death. This review provides an overview of the role of mitochondrial oxidative stress in podocytopathies and discusses its role in the cell death of the podocyte, aiming to identify the novel targets to improve the treatment of patients with podocytopathies.

MAD2B-mediated cell cycle reentry of podocytes is involved in the pathogenesis of FSGS.

Rationale: Focal segmental glomerulosclerosis (FSGS) is characterized by the dysfunction of "post-mitotic" podocytes. The reentry of podocytes in the cell cycle will ultimately result in cell death. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of anaphase-promoting complex (APC)/cyclosome, precisely controls the metaphase to anaphase transition and ordered cell cycle progression. However, the role of MAD2B in FSGS podocyte injury remains unknown. Methods: To explore MAD2B function in podocyte cell cycle reentry, we used conditional mutant mice lacking MAD2B selectively in podocytes in ADR-induced FSGS murine model. Additionally, KU-55933, a specific inhibitor of ataxia-telangiectasia mutated (ATM) was utilized in vivo and in vitro to explore the role of ATM in regulating MAD2B. Results: The expression of MAD2B in podocytes was dramatically increased in patients with FSGS and ADR-treated mice along with podocyte cell cycle reentry. Podocyte-specific knockout of MAD2B effectively attenuated proteinuria, podocyte injury, and prevented the aberrant cell cycle reentry. By bioinformatics analysis we revealed that ATM kinase is a key upstream regulator of MAD2B. Furthermore, inhibition of ATM kinase abolished MAD2B-driven cell cycle reentry and alleviated podocyte impairment in FSGS murine model. In vitro studies by site-directed mutagenesis and immunoprecipitation we revealed ATM phosphorylated MAD2B and consequently hampered the ubiquitination of MAD2B in a phosphorylation-dependent manner. Conclusions: ATM kinase-MAD2B axis importantly contributes to the cell cycle reentry of podocytes, which is a novel pathogenic mechanism of FSGS, and may shed light on the development of its therapeutic approaches.

Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System.

BACKGROUND: Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. METHODS: In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. RESULTS: The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. CONCLUSION: Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

The Role of Long-Lived Plasma Cells in Antibody-Mediated Rejection of Kidney Transplantation: An Update.

BACKGROUND: Antibody-mediated rejection (ABMR) following kidney transplant is closely associated with poor prognosis of the recipients. Long-lived plasma cells (LLPCs) produce alloantibodies as long as life time and play a crucial role in ABMR. SUMMARY: LLPCs generate from germinal centers and reside in survival niches in the bone marrow as well as the inflamed tissues. They are the main and long-term source of the antibodies. LLPCs mediate ABMR via the generation of preformed antibodies in sensitized patients and de novo antibodies after transplantation. They have been acknowledged as the leading causes of ABMR; however, LLPCs are insensitive to traditional immunosuppressive therapy that removes B cells. Strategies targeting LLPCs, such as antithymocyte globulin, proteasome inhibitors as well as monoclonal antibodies, are promising methods to persistently and thoroughly clear the entire PC pool. KEY MESSAGE: LLPCs play an important role in ABMR by producing alloantibodies continually, and targeting LLPCs might be a novel and effective approach against ABMR.

Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for poly(3-hydroxybutyrate) (PHB) production.

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for ß-ketothiolase (phaA Cma , 1179 bp), acetoacetyl-CoA reductase (phaB Cma , 738 bp), and PHA synthase (phaC Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD600, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.

Combination of low-energy shock-wave therapy and bone marrow mesenchymal stem cell transplantation to improve the erectile function of diabetic rats.

Stem cell transplantation and low-energy shock-wave therapy (LESWT) have emerged as potential and effective treatment protocols for diabetic erectile dysfunction. During the tracking of transplanted stem cells in diabetic erectile dysfunction models, the number of visible stem cells was rather low and decreased quickly. LESWT could recruit endogenous stem cells to the cavernous body and improve the microenvironment in diabetic cavernous tissue. Thus, we deduced that LESWT might benefit transplanted stem cell survival and improve the effects of stem cell transplantation. In this research, 42 streptozotocin-induced diabetic rats were randomized into four groups: the diabetic group (n = 6), the LESWT group (n = 6), the bone marrow-derived mesenchymal stem cell (BMSC) transplantation group (n = 15), and the combination of LESWT and BMSC transplantation group (n = 15). One and three days after BMSC transplantation, three rats were randomly chosen to observe the survival numbers of BMSCs in the cavernous body. Four weeks after BMSC transplantation, the following parameters were assessed: the surviving number of transplanted BMSCs in the cavernous tissue, erectile function, real-time polymerase chain reaction, and penile immunohistochemical assessment. Our research found that LESWT favored the survival of transplanted BMSCs in the cavernous body, which might be related to increased stromal cell-derived factor-1 expression and the enhancement of angiogenesis in the diabetic cavernous tissue. The combination of LESWT and BMSC transplantation could improve the erectile function of diabetic erectile function rats more effectively than LESWT or BMSC transplantation performed alone.

Corrigendum to "Combination of low-energy shock-wave therapy and bone marrow mesenchymal stem cell transplantation to improve the erectile function of diabetic rats" by Hai-Tao Shan et al.

[This corrects the article DOI: 10.4103/1008-682X.184271.].

The Feasibility of Thermophilic Caldimonas manganoxidans as a Platform for Efficient PHB Production.

Recently, poly(3-hydroxybutyrate) (PHB) has been found in a few thermophilic strains where several advantages can be gained from running fermentation at high temperatures. Caldimonas manganoxidans, a thermophilic gram-negative bacterium, was investigated for the feasibility as a PHB-producing strain. It is suggested that the best fermentation strategy for achieving the highest PHB concentration of 5.4 ± 1.1 g/L (from 20 g/L glucose) in 24 h is to use the fermentation conditions that are favored for the bacterial growth, yet temperature and pH should be chosen at conditions that are favored for the PHB content. Besides, the above fermentation conditions produce PHB that has a high molecular weight of 1274 kDa with a low polydispersity index (PDI) of 1.45, where the highest Mw of PHB of 1399 kDa (PDI of 1.32) is obtained in this study. To the best knowledge of authors, C. manganoxidans has the best PHB productivity among the thermophiles and is comparable to those common PHB-producing mesophiles.

[The effect of carvedilol on coronary flow reserve in patients with dilated cardiomyopathy].

OBJECTIVE: To investigate the change of coronary flow reserve (CFR) in patients with dilated cardiomyopathy (DCM) with non-invasive transthoracic stress echocardiography before and after administration of carvedilol. METHODS: Seventy-five patients with DCM were included and divided into a group with heart failure (HF), a group without (non HF) and 30 healthy subjects with normal angiography and negative ECG exercise test were served as controls. In addition to traditional treatment, all patients were given enough carvedilol in 6 months. Doppler measurement of distal left anterior descending was recorded at rest and hyperemic state after adenosine infusion and CFR was calculated before and after the treatment. RESULTS: Compared with the controls, HF group and non HF group had greater left atrial diameter (LAd) and left ventricular diastolic diameter (LVDd) but less left ventricular ejection fraction (LVEF) and E/A than the controls before treatment (P < 0.05). LAd, LVDd and LVEF of the HF group and non HF group improved after treatment and there was significant difference of these indexes between the two groups (P < 0.05). Compared with the controls, HF group and non HF group had lower CFR before treatment (2.35 +/- 0.28 vs 2.57 +/- 0.31 vs 3.20 +/- 0.29, P < 0.05). After treatment with carvedilol, CFR rised in these two groups. Although CFR was still lower in the HF group than that in the control group (2.68 +/- 0.30 vs 3.20 +/- 0.29, P < 0.05), there was no difference between the non HF group and the controls (3.13 +/- 0.36 vs 3.20 +/- 0.29, P > 0.05). CONCLUSIONS: CFR decreased in patients with DCM and patients with heart failure had lower CFR than those without. Carvedilol could not only reverse the ventricular remodeling due to DCM, but also improve CFR in those patients. Detection of CFR with stress echocardiography could evaluate the effect of carvedilol earlier than the index of traditional echocardiography.

In vitro effects of erythromycin on RANKL and nuclear factor-kappa B by human TNF-alpha stimulated Jurkat cells.

The receptor activator of NF-kappaB ligand (RANKL) and its signal downstream nuclear factor-kappaB (NF-kappaB) are critical regulators for immune responses as well as bone remodeling. The present study aimed to examine the effects of erythromycin (EM) on the activation of RANKL, correlation with NF-kappaB expression, proliferation and apoptosis of human Jurkat T cells. Jurkat T cells were pretreated with 100 pmol/l tumor necrosis factor-alpha (TNF-alpha) for 1 h followed by various concentrations of EM for 24 h. The mRNA expressions of RANKL and NF-kappaB were examined by RT-PCR. The protein expression of NF-kappaB was analyzed by Western blot. The protein level of RANKL was examined by flow cytometry, immunofluorescence microscopy and Western blot analyses. We also examined proliferation of Jurkat T cells by MTT assay, apoptosis by flow cytometry analysis after staining with PI and morphological observation after AO/EB staining. The results showed that EM inhibited TNF-alpha-induced expressions of RANKL and NF-kappaB at both mRNA and protein levels in a concentration-dependent manner. The expression of RANKL was correlated with the expression of NF-kappaB. Moreover, EM influenced the proliferation and apoptosis of human Jurkat T cells. These data suggest that EM acts as an anti-inflammatory agent not only to interact with the expression of NF-kappaB and the proliferation of human Jurkat T cells, but also to reduce the level of RANKL.

Establishment of a transplantable human myeloid BALB/c nude mouse model.

To establish a mouse model bearing transplantable human chronic myeloid leukemia for hematopoietic stem cell transplantation to treat leukemia, 4 - 5-week-old female BALB/c nude mice were given cyclophosphamide 2 mg/mouse at day -2, -1, and then the human chronic myeloid leukemia K562 cells were engrafted into the mice at day 0 by injection via tail vein or peritoneal cavity. PB and BM cells were collected, the CD45, CD13, and CD33 antigens were delected by using FCM, the bcr/abl fusion gene mRNA was examined by RT-PCR. The results showed that transplantable leukemic mice could be yielded from 4 - 5-week-old nude mice either by injection through tail vein or peritoneal cavity when the total number of inoculated tumor cells was more than 2 x 10(5) per mouse, whether being pretreated with 2 mg CTX/mouse or not. The transplanted mice could survive 30 - 60 day with leukemia. In conclusion, the mouse model bearing leukemia can be established by inoculation 2 x 10(5) K562 cells into immunodeficient BALB/c nude mice.