One-tube nested MGB Probe Real-time PCR assay for detection of Echinococcus multilocularis infection in plasma cell free DNA.

INTRODUCTION: The main objective of this study was to develop a One-tube nested MGB probe real-time PCR Assay for detecting Echinococcus multilocularis infection in human plasma cell free DNA (cfDNA). METHODS: cfDNA was extracted from 10 E.m.-infected patients using a NucleoSnap DNA Plasma Kit and characterized by genomic sequencing. We designed nested PCR primers and MGB probe for Echinococcus multilocularis detection. The specificity, sensitivity and reproducibility of this assay were analyzed, and its validity was confirmed in 13 early stage clinical samples. RESULTS: Several Echinococcus multilocularis-specific sequences were detected in the cfDNA of E.m.-infected patients, and CBLO020001206.1 was selected as the candidate sequence. We designed the primers and probe for the one tube nested real-time PCR. No cross-reactions with E.g. were observed. The detection limit was as low as 1 copy for Echinococcus multilocularis. The coefficients of variation were lower than 5% in intra- and inter-assays. 11 out of 13 patients were positive with nested MGB Probe PCR Assay and 3 patients were positive without outer primer in early stage Alveolar Echinococcosis pateints. CONCLUSION: The one-tube nested MGB probe real-time PCR assay is a simple, rapid, and cost-effective method for detection of Echinococcus multilocularis infection in patients' Plasma DNA.

CLINICAL SIGNIFICANCE AND CORRELATION ANALYSIS OF SERUM FERRITIN IN PATIENTS WITH HEPATIC ALVEOLAR ECHINOCOCCOSIS.

The serum ferritin (SF) levels of patients with hepatic alveolar echinococcosis (HAE) were compared to the laboratory reference value, and the correlation between SF and associated parameters in patients with HAE was assessed. Hematological and imaging data of 245 patients with HAE were collected. Patients were classified into the LSF group (SF ≤ 204 ng/ml) or HSF group (SF > 204 ng/ml) according to the level of SF. There was no significant difference in the serum iron level between groups (P > 0.05). Significant differences in unsaturated iron-binding capacity (UIBC), liver function, blood coagulation, lipid, blood cell count, and lesion characteristics were observed (P < 0.05). Correlation analysis showed that SF was related to UIBC, γ-glutamyl transferase, total bilirubin (TBIL), direct bilirubin (DBIL), fibrinogen (FIB), neutrophil count, and maximal lesion diameter (all absolute rs ≥ 0.4). The correlation coefficient between SF and UIBC showed the highest absolute value (rs = -0.556, P < 0.001). Single-factor linear regression analysis showed that TBIL and DBIL showed the R2 values were 0.221 and 0.220, and the R2 values of UIBC, FIB, and maximal lesion diameter were 0.157, 0.174, and 0.167, respectively, and those of the remaining indicators were <0.1. Multi-factor binary logistic regression analysis showed that UIBC (P < 0.001, OR = 0.909), FIB (P = 0.020, OR = 1.662), hemoglobin (HGB) (P = 0.002, OR = 1.029), and maximal lesion diameter (P = 0.002, OR = 1.146) were significant factors influencing SF abnormalities. SF levels in some patients with HAE were higher than the laboratory reference value. Correlation and regression analysis of SF suggested that the UIBC, FIB, HGB, and maximal lesion diameter were related to SF and affected the SF level. These results may be helpful for the diagnosis and severity assessment of HAE in the future.

Primary splenic hydatidosis: a case report.

We herein report a case of primary splenic hydatidosis to provide data regarding the diagnosis, treatment, and epidemiological statistics of this disease. The patient was from a pastoral area and was diagnosed with primary splenic hydatidosis with chronic atrophic gastritis. The patient had no history of surgical treatment of hydatidosis. The diagnosis was mainly based on possible exposure to endemic areas, imaging findings, serological test results, and operative and pathological examination findings. Laparoscopic splenectomy was performed, and regular albendazole therapy was given after the operation. The patient was admitted to the hospital for gastrointestinal bleeding 3 months postoperatively, and she was successfully treated and discharged. No recurrence of hydatid foci has been observed since the follow-up.

Effects of transcription factor EB on oxidative stress and apoptosis induced by high glucose in podocytes.

The aim of the present study was to investigate the effects of transcription factor EB (TFEB) overexpression on oxidative stress, mitochondrial function and apoptosis in podocytes induced with high glucose. High glucose­induced time­dependent changes in TFEB expression were identified and nuclear translocation of TFEB was observed in podocytes. Overexpression of TFEB markedly reduced high glucose­induced oxidative stress in podocytes, and increased the expression of superoxide dismutase 2 and heme oxygenase 1 antioxidant enzymes. It was further observed that TFEB overexpression could partially restore the expression of peroxisome proliferator­activated receptor­Î³ coactivator­1α, transcription factor A, mitochondrial, and cytochrome c oxidase subunit 4, thereby enhancing mitochondrial biosynthesis. Furthermore, overexpression of TFEB reduced mitochondrial swelling and fragmentation, restored mitochondrial membrane potential, and contributed to the restoration of mitochondrial function. By overexpressing TFEB, it was revealed that TFEB increased the ratios of phosphorylated (p)­Akt/Akt and p­Bad/Bad, and the expression of downstream Bcl­xl, and reduced the ratio of Bax/Bcl­2 and the expression of cleaved­caspase­3 compared with high glucose­treatment. Furthermore, when the Akt phosphorylation inhibitor Ly294002 was added, the improvement by TFEB to high glucose­induced apoptosis was significantly reduced. These findings suggest that overexpressing TFEB could reduce the production of reactive oxygen species in podocytes in a high glucose environment, relieve oxidative stress, promote mitochondrial biogenesis and renewal functions, and reduce high glucose­induced podocyte apoptosis by activating the Akt/Bad pathway.

Magnetic resonance imaging evaluation of characteristics of vascular invasion in intermediate and advanced hepatic alveolar echinococcosis.

The objective of the present study was to provide a basis for the personalized treatment of intermediate and advanced hepatic alveolar echinococcosis (HAE) by elucidating the characteristics of vascular invasion and lesion growth. A total of 160 patients with intermediate and advanced HAE who were subjected to plain as well as contrast-enhanced 3.0-T magnetic resonance imaging prior to surgery were analyzed. Pathological and intra-operative observations of the subjects were also considered. The size and location of HAE lesions, vascular invasion characteristics and growth patterns were assessed. A total of 78 patients (48.75%) had lesions involving the S5-8 segment/partial right liver lobe, 21 (13.13%) had involvement in the S2-4 segment/partial left liver lobe and 61 (38.13%) had lesions that transcended the left and right liver lobes. Pathological examination revealed that the vascular invasion rates of the hepatic portal veins, intrahepatic veins (left, central and right vein, and inferior vena cava) and hepatic arteries were 51.88, 43.28 and 26.87%, respectively. Liver hilum invasion was observed in 128 patients (80.00%), 71 of which (44.38%) presented with invasion of the primary porta hepatis, 11 (6.88%) with invasion of the secondary porta hepatis and 46 (28.75%) with invasion of the primary as well as the secondary porta hepatis. In conclusion, the growth pattern of intermediate and advanced HAE is determined by the site, blood supply and activity of the lesion. The current study demonstrated that lesions tend to invade the intrahepatic venous system and porta hepatis, and to target veins rather than arteries.

Resveratrol ameliorates podocyte damage in diabetic mice via SIRT1/PGC-1α mediated attenuation of mitochondrial oxidative stress.

Excessive generation of mitochondrial reactive oxygen species (ROS) is considered to be initiating event in the development of diabetic nephropathy (DN). Mitochondrial biosynthesis mediated by coactivator PGC-1α and its downstream transcription factors NRF1 and TFAM may be a key target in maintaining mitochondrial function. Resveratrol (RESV), a natural polyphenolic antioxidant, is a potent SIRT1 agonist. In this study we established diabetes mouse and podocyte exposed to high glucose as in vivo and in vitro models to investigate the efficacy and mechanism of RESV on renoprotection. We found that RESV alleviated proteinuria of diabetic mice, decreased malondialdehyde content while increased Mn-SOD activity in renal cortex, inhibited the apoptosis of glomerular podocytes and renal tubular epithelial cells, ameliorated pathological manifestations, and restored the expression of SIRT1 and PGC-1α in renal tissues of DN mice. In podocytes exposed to high glucose, RESV inhibited excessive ROS production and apoptosis. In addition, RESV decreased mitochondrial ROS production, improved respiratory chain complex I and III activity, elevated mitochondrial membrane potential, and inhibited the release of Cyto C and Diablo in the mitochondria into the cytoplasm. Taken together, our findings suggest that RESV ameliorates podocyte damage in diabetic mice via SIRT1/PGC-1α mediated attenuation of mitochondrial oxidative stress.