Assessment of mitochondrial function in metabolic dysfunction-associated fatty liver disease using obese mouse models.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is characterized by deregulated hepatic lipid metabolism; however, the association between MAFLD development and mitochondrial dysfunction has yet to be confirmed. Herein, we employed high-resolution respirometry, blue native polyacrylamide gel electrophoresis-based in-gel activity measurement and immunoblot analysis to assess mitochondrial function in obesity-induced mouse models with varying degrees of MAFLD. Results showed a slight but significant decrease in hepatic mitochondrial respiration in some MAFLD mice compared to mice fed a standard diet. However, the activities and levels of mitochondrial oxidative phosphorylation complexes remained unchanged during obesity-induced MAFLD progression. These results suggest that mitochondrial function, particularly oxidative phosphorylation, was mildly affected during obesity-induced MAFLD development. Moreover, transcriptome profiling of mouse and human liver tissues with varying degrees of MAFLD revealed that the decreased activation of mitochondria-related pathways was only associated with MAFLD of a high histological grade, whereas the major regulators of mitochondrial biogenesis were not altered in mice or humans during MAFLD development. Collectively, our results suggest that impaired hepatic mitochondrial function is not closely associated with obesity-induced MAFLD. Therefore, therapeutic strategies targeting mitochondria for the treatment of MAFLD should be reconsidered.

A simple and rapid protein purification method based on cell-surface display of SUMO-fused recombinant protein and Ulp1 protease.

The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.

Redox regulation by SOD2 modulates colorectal cancer tumorigenesis through AMPK-mediated energy metabolism.

Colorectal cancer (CRC) is a common malignancy. Many reports have implicated aberrant mitochondrial activity in the progression of CRC, with particular emphasis on the dysregulation of redox signaling and oxidative stress. In this study, we focused on manganese superoxide dismutase (MnSOD/SOD2), a key antioxidant enzyme, which maintains intracellular redox homeostasis. Current literature presents conflicting mechanisms for how SOD2 influences tumorigenesis and tumor progression. Here, we explored the role of SOD2 in CRC specifically. We found high levels of SOD2 expression in CRC tissues. We carried out a series of experiments to determine whether knockdown of SOD2 expression in CRC cell lines would reverse features of tumorigenesis. We found that reduced SOD2 expression decreased cell proliferation, migration, and invasion activity in CRC cells. Results from an additional series of experiments on mitochondrial function implicated a dual role for SOD2 in promoting CRC progression. First, proper level of SOD2 helped CRC cells maintain mitochondrial function by disposal of superoxide (O2.- ). Second, over-expression of SOD2 induced H2 O2 -mediated tumorigenesis by upregulating AMPK and glycolysis. Our results indicate that SOD2 may promote the occurrence and development of CRC by regulating the energy metabolism mediated by AMPK signaling pathways.

Efficacy and safety of thrombopoietin receptor agonists in children with chronic immune thrombocytopenia: a meta-analysis.

BACKGROUND AND AIM: Thrombopoietin receptor agonists (TPO-RAs) have been shown to be safe and effective for adults with chronic immune thrombocytopenia (ITP). The aim of this meta-analysis is to assess the efficacy and safety of thrombopoietin receptor agonists for children with chronic ITP. MATERIALS AND METHODS: Clinical randomized controlled trials (RCTs) evaluating the efficacy and safety of TPO-RAs in pediatric ITP patients published up to June 2017 were retrieved from PubMed, Cochrane Library, and Embase databases. Relevant data were extracted, and the Physiotherapy Evidence Database scale was used to assess the methodological quality. Stata/SE 12.0 was used to perform a meta-analysis. RESULTS: Seven RCTs were included, with 238 patients and 107 patients in the TPO-RA group and the control group, respectively. Assessing efficacy, better results were found in the TPO-RA group for the rate of overall platelet response, durable response, and rescue medication needed. Furthermore, the TPO-RA group yielded superior results in the incidence of clinically significant bleeding events but had a comparable result in the incidence of any bleeding events and severe bleeding events. No significant difference was found between the two groups in health-related quality of life and parental burden. Assessing safety, no significant difference was found between the two groups in the incidence of any adverse events and severe adverse events. CONCLUSIONS: TPO-RAs are effective and safe agents for the treatment of chronic ITP in pediatric patients. Eltrombopag appears to be better than romiplostim in terms of the rate of rescue medication needed and clinically significant bleeding events.

[Detection of lymph node micrometastasis for patients with extrahepatic cholangiocarcinoma and its prognostic significance].

OBJECTIVE: To observe the expression of antibodies of cytokeratin 19 and 20 in lymph node micrometastasis in patients with extrahepatic cholangiocarcinoma (EHCC), evaluate the prognostic significance of lymph node (LN) micrometastasis and study the correlation between lymph node micrometastasis and clinicopathological features, CA19-9 and CEA. METHODS: A total of 279 lymph nodes was intra-operatively collected from 59 EHCC patients and routine histological examination performed. Immunohistochemical staining was performed on all samples by the murine antibodies of anti-CK19 and anti-CK20 respectively. Then the micrometastasis was identified microscopically according to the color of cells. The results were analyzed according to clinical, pathological and follow-up data. And the relation of micrometastasis with clinical pathological factors and its impact upon survival rate were analyzed. RESULTS: Among 59 EHCC patients, 14 (23.72%) LN metastasis were found with HE staining and 21 micrometastases with CK staining. The incidence of nodal involvement in 59 EHCC patients increased from 5.37% (15/279) by HE staining to 13.98% (39/279) by CK staining. Among 45 patients not positive for LN metastases with HE staining, CK staining was positive in 7 patients and the incidence of micrometastasis was 15.56%. The preoperative serum CA19-9 levels in patients with LN micrometastasis was higher than that those without LN metastasis (P < 0.05). And there was a positive correlation between occult nodal micrometastasis and serum concentrations of CA19-9 (r(s) = 0.371, P < 0.05). The histological type and lymphatic vessel infiltration of tumor were the most importance factors for LN micrometastasis through Logistic regression analysis (P < 0.05). CONCLUSION: The CK immunohistochemical staining can detect the micrometastases in HE negative LN. And LN micrometastasis can more accurately predict the prognosis of EHCC patients.

[Expression of Survivin protein in extrahepatic cholangiocarcinoma and its relationship with the prognosis].

OBJECTIVES: To investigate the expression of Survivin in patients with extrahepatic cholangiocarcinoma (EHCC) and its relationship with clinicopathological features of EHCC, and the correlation between the expression of Survivin and lymph node micrometastasis, tumor markers, and the prognosis of EHCC. METHODS: The expression of Survivin protein in paraffin-embedded specimens of 59 patients with EHCC and their 20 para-carcinoma tissues were evaluated by S-P method of immunohistochemical staining. The correlation between the expression of Survivin and the lymph node micrometastasis, clinicopathological features of EHCC and the prognosis of EHCC were analyzed. RESULTS: The positive expression rate of Survivin protein was 67.8% (40/59) in paraffin-embedded specimens of 59 patients with EHCC and was 20.0% (4/20) in para-carcinoma tissues, and difference between carcinoma tissues and para-carcinoma tissues was significant (P<0.01). Histological differentiation in EHCC had a negative correlation with the expression of Survivin protein, while the expression of Survivin protein in EHCC had a positive correlation with TNM of EHCC, lymphatic vessel infiltration, lymph node metastasis and perineural invasion (P<0.05). The serum CA19-9 levels in the positive group with expression of Survivin protein was (290,300+/-55 500) U/L and was obviously higher than that in the negative group [(113,300+/-31,400) U/L, P<0.05]. The mean survival time of the patients with negative expression of Survivin protein was higher than that of the patients with positive expression (43.5 vs. 21.1 months, P<0.01). Screened to significance univariate, the multivariate analysis through Cox proportional hazard model analysis showed that lymph node metastasis, residual tumor margins, and expression of Survivin protein were independent prognosis factors of the patients with EHCC (P<0.05, P<0.01, P<0.01). CONCLUSIONS: The expression of Survivin protein in EHCC has a negative correlation with histological differentiation, while has a positive correlation with lymphatic vessel infiltration and serum CA19-9 concentrations. The expression of Survivin protein maybe an independent prognosis factor of the patients with EHCC.