Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes. METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms. RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

4-Octyl itaconate inhibits poly(I:C)-induced interferon-ß secretion in mouse bone marrow-derived macrophages partially by activating Nrf2.

Viruses have become a major threat to human health. Interferon-ß (IFN-ß) has a key role in the antivirus process, as it can increase the expression of antivirus-associated genes. Itaconate and its derivatives can regulate the immune response, secretion of inflammatory factors, and pyroptosis of macrophages. The effect of itaconate on IFN-ß secretion of double-stranded RNA-induced macrophages are not well known. A derivative of itaconate, 4-octoyl itaconate (4-OI), was used to treat mouse bone marrow-derived macrophages (BMDM) induced with 100 µg/mL poly(I:C). The IFN-ß concentration was detected through ELISA, and IFN-ß mRNA expression was detected through quantitative PCR. High-throughput transcriptome sequencing was used to analyze changes in the BMDM transcriptome after 4-OI treatment. The Nrf2 expression was knocked down with siRNA.4-OI inhibited poly(I:C)-induced IFN-ß secretion and mRNA expression in BMDM. Results of transcriptome sequencing revealed that 4-OI downregulated 1047 genes and upregulated 822 genes. GO and KEGG enrichment of differently expressed genes revealed that many downregulated genes were related to the anti-virus process, whereas many upregulated genes were related to metabolism. The Nrf2 inhibitor ML385 and Nrf2 siRNA could partially reverse the inhibitory effect of 4-OI. In conclusion, 4-octyl itaconate could inhibit the poly(I:C)-induced interferon-ß secretion in BMDM partially by regulating Nrf2.

The value of Sonoclot detection technology to guide the clinical medication of the perioperative anticoagulation and antiplatelet therapy in patients with acute myocardial infarction undergoing emergent PCI.

The value of Sonoclot detection technology to guide the clinical medication of the perioperative anticoagulation and antiplatelet therapy in patients with acute myocardial infarction (AMI) undergoing emergent percutaneous coronary intervention (PCI) was estimated. One hundred and twenty-eight patients were randomly divided into control group and observation group with 64 cases in each group. Control group adopted routine blood coagulation indexes, including prothrombin time, activated partial thromboplastin time, fibrinogen and plasma thrombin time, platelet count and platelet aggregation turbidity analysis; observation group adopted Sonoclot detection technology, including activated clotting time, coagulation rate and platelet function. Anticoagulant therapy selected was of low molecular weight heparin calcium perioperatively, intraoperative unfractionated heparin, and clopidogrel (75 mg) combined with aspirin enteric-coated tablets (100 mg) as antiplatelet drugs. The therapy was administered in accordance with blood coagulation results. The blood coagulation time, postoperative creatine kinase isoenzyme MB, cardiac troponin I and B-type natriuretic peptide levels in the observation group are significantly lower than those in the control group (P<0.05) though the operating time and specifications of the stenting did not show any significant difference (P>0.05). The incidence of recurrent myocardial infarction, microembolism, acute and subacute thrombosis and bleeding events in the observation group are significantly lower than those in the control group (P<0.05). In the control group, there is no difference in the coagulation indexes of the patients with thrombosis events or bleeding events or no event (P>0.05). Whereas, in the observation group, there is significant difference in coagulation indexes of the patients with thrombosis events or bleeding events or no event (P<0.05). In conclusion, Sonoclot detection technology instructs emergent PCI treatment in AMI patients to shorten the detection time of blood coagulation, reduce the degree of myocardial injury, reduce the incidence of perioperative thrombosis and bleeding events. Furthermore, it has great value in guiding the clinical medication of anticoagulation and antiplatelet therapy.

Surgical Incision and Approach in Thoracolumbar Extreme Lateral Interbody Fusion Surgery: An Anatomic Study of the Diaphragmatic Attachments.

STUDY DESIGN: Cadaveric study. OBJECTIVE: To provide anatomical basis for deciding the surgical approach and skin incision in thoracolumbar extreme lateral interbody fusion (XLIF) by delineating the attachment points of diaphragm. SUMMARY OF BACKGROUND DATA: Although the general anatomy of the thoracic diaphragm is well described, the specific attachment points of diaphragm concerned with the XLIF approach is yet to be elaborated. METHODS: Dissections were performed on 21 cases of formalin fixed specimens (12 males, 9 females, a total of 42 sets of data). Special attention was paid to the attachment points of diaphragm on both sides at the midaxillary line (MAL point) and the vertebral level parallel to the MAL point (VL-MAL). The attachment points of diaphragm on the front and back edge of the spinal column (FES point and BES point) were also described. RESULTS: The MAL point of diaphragm muscle lied between the inferior edge of the 10th rib and the superior edge of the 12th rib (20 out of 21 on left, 21 out of 21 on right). VL-MAL lied between L1 and L2 vertebrae level (20 out of 21 on left, 18 out of 21 on right). The attachments on both sides of the vertebral column mainly located between the upper edge of T12 vertebrae and L1-L2 disc (38 out of 42). CONCLUSION: A transthoracic approach should be considered when the target level was above T12 vertebrae, whereas a retroperitoneal approach should be chosen when target level was below L1-L2 disc. If the target level is located between T12 and L1-L2 disc, whether via transthoracic, retropleural, or retroperitoneal approach should be determined according to the conditions of patients and the skill and experience of the surgeon. Incision should be made above the 10th rib for the transthoracic approach and below the 12th rib for the retroperitoneal approach. LEVEL OF EVIDENCE: 4.

Curdione inhibits proliferation of MCF-7 cells by inducing apoptosis.

BACKGROUND: Curdione, one of the major components of Curcuma zedoaria, has been reported to possess various biological activities. It thus might be a candidate anti-flammatory and cancer chemopreventive agent. However, the precise molecular mechanisms of action of curdione on cancer cells are still unclear. In this study, we investigated the effect of curdione on breast cancer. MATERIALS AND METHODS: Xenograft nude mice were used to detect the effect of curdione on breast cancer in vivo; we also tested the effect of curdione on breast cancer in vitro by MTT, Flow cytometry, JC-I assay, and western blot. RESULTS: Firstly, we found that curdione significantly suppressed tumor growth in a xenograft nude mouse breast tumor model in a dose-dependent manner. In addition, curdione treatment inhibited cell proliferation and induced cell apoptosis. Moreover, after curdione treatment, increase of impaired mitochondrial membrane potential occurred in a concentration dependent manner. Furthermore, the expression of apoptosis-related proteins including cleaved caspase-3, caspase-9 and Bax was increased in curdione treatment groups, while the expression of the anti-apoptotic Bcl-2 was decreased. Inhibitors of caspase-3 were used to confirm that curdione induced apoptosis. CONCLUSIONS: Overall, our observations first suggested that curdione inhibited the proliferation of breast cancer cells by inducing apoptosis. These results might provide some molecular basis for the anti-cancer activity of curdione.

[Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene].

OBJECTIVE: To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene. METHODS: Based on the human ILK gene sequences, RNAi target sequences were designed and cloned into the lentiviral vector pSicoR-eGFP by restriction endonuclease HpaI and XhoI double digestion and T4 DNA ligase ligation. Based on the human mda7 gene sequences, PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro. After the candidate clones were identified by DNA sequencing, the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles. Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector. The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot, respectively. The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively. RESULTS: ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed. Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector. The transfection efficiency of the collected virus exceeded 90% in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency. The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly. The mda7-pLVX-Puro lentiviral vector increased the expression of mda7 in PC-3 cells, and the ability was maintained for one month. Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells (Ps<0.05). CONCLUSION: The lentiviral vectors of ILK knockdown and mda7 over-expression have been successfully constructed and identified. The recombinant lentivirus can efficiently infect human prostate cancer PC-3 cells, in which ILK expression is inhibited and mda7 is over-expressed.

Gambogenic acid induction of apoptosis in a breast cancer cell line.

BACKGROUND: Gambogenic acid is a major active compound of gamboge which exudes from the Garcinia hanburyi tree. Gambogenic acid anti-cancer activity in vitro has been reported in several studies, including an A549 nude mouse model. However, the mechanisms of action remain unclear. METHODS: We used nude mouse models to detect the effect of gambogenic acid on breast tumors, analyzing expression of apoptosis-related proteins in vivo by Western blotting. Effects on cell proliferation, apoptosis and apoptosis-related proteins in MDA-MB-231 cells were detected by MTT, flow cytometry and Western blotting. Inhibitors of caspase-3,-8,-9 were also used to detect effects on caspase family members. RESULTS: We found that gambogenic acid suppressed breast tumor growth in vivo, in association with increased expression of Fas and cleaved caspase-3,-8,-9 and bax, as well as decrease in the anti-apoptotic protein bcl-2. Gambogenic acid inhibited cell proliferation and induced cell apoptosis in a concentration-dependent manner. CONCLUSION: Our observations suggested that Gambogenic acid suppressed breast cancer MDA-MB-231 cell growth by mediating apoptosis through death receptor and mitochondrial pathways in vivo and in vitro.

Prognostic significance of serum miRNA-21 expression in human non-small cell lung cancer.

BACKGROUND: Deregulation of microRNAs (miRNAs) plays important roles in tumor progression. The aim of this study was to investigate miR-21 expression in serum of non-small cell lung cancer (NSCLC) and its correlation with prognosis of NSCLC patients. METHODS: Dysregulated miRNAs in NSCLC serum were identified by microarray. MiR-21 expression in NSCLC and control serum was detected by TaqMan RT-PCR assay. The correlation of serum miR-21 with clinicopathological factors of NSCLC patients was analyzed. Furthermore, the prognostic significance of serum miR-21 was analyzed by using Kaplan-Meier curves with log-rank tests and a Cox proportional hazard model. RESULTS: The level of miR-21 expression was higher in NSCLC serum samples than in control serum samples (P < 0.01). High serum miR-21 was significantly correlated with tumor-node metastases stage and lymph node metastasis of NSCLC patients (P = 0.016 and 0.026, respectively). The 3-year actuarial overall survival rates in NSCLC patients with high serum miR-21 expression (39.8%) was significantly shorter than those with low serum miR-21 expression (58.2%; P < 0.001). Furthermore, univariate and multivariate analyses for overall survival showed that serum miR-21 expression was an independent prognostic factor for NSCLC patients (P = 0.015, RR = 2.01, 95% CI: 1.78-3.26). CONCLUSION: Serum miR-21 expression might be useful as a prognostic marker for NSCLC patients.

Functional and biological analysis of Bcl-xL expression in human osteosarcoma.

Bcl-xL, a member of Bcl-2 protein family functioned as dominant regulators of apoptotic cell death, has been reported to play important roles in malignant transformation and tumor development. In the present study, our aim was to explore the roles of Bcl-xL overexpression and determine its possibility as a therapeutic target in human osteosarcoma. Real-time quantitative RT-PCR and Western blot or immunohistochemistry assays were performed to detect the expression of Bcl-xL mRNA and protein in human osteosarcoma cell lines or tissue samples. The expression of other Bcl-2 family proteins (Bcl-2, Mcl-1, Bim and Bik) in osteosarcoma tissues was also detected by immunohistochemistry. The associations of Bcl-xL mRNA expression with clinicopathologic factors and prognosis of osteosarcoma patients were evaluated. RNA interference or gene overexpression technologies were employed to downregulate or upregulate endogenous Bcl-xL expression in osteosarcoma cells and the effects of Bcl-xL downregulation or upregulation on phenotypes and chemo- or radiosensitivity of human osteosarcoma cells were analyzed. Finally, the mechanism of synergistic effects of Bcl-xL downregulation and chemo- or radiotherapy was explored by detecting the activity of caspase-3. The expression levels of Bcl-xL mRNA and protein in high metastatic osteosarcoma cells showed higher than those in low metastatic osteosarcoma cells. Moreover, the levels of Bcl-xL mRNA expression were significantly higher in osteosarcoma tissues than those in chondroma or corresponding non-tumor tissues (P<0.01), and osteosarcoma tissues showed stronger immunostaining of Bcl-xL protein than non-tumor tissues. The stronger staining of Bcl-2 and Mcl-1 proteins was also observed, while the staining of pro-apoptotic proteins (Bim and Bik) was significantly weaker or not detected in osteosarcoma tissues. The higher levels of Bcl-xL mRNA expression were significantly correlated with advanced clinical stage (P=0.005) or hematogenous metastasis (P=0.001) of osteosarcoma patients. Osteosarcoma patients with higher Bcl-xL mRNA expression showed a poorer survival compared with those with lower expression (P=0.039). Bcl-xL downregulation or upregulation could significantly reduce or increase the proliferation capacity of osteosarcoma cells. Furthermore, Bcl-xL downregulation could significantly enhance in vitro chemo- or radiosensitivity of osteosarcoma cells, which might be associated with elevated activity of caspase-3. Taken together, overexpression of Bcl-xL may play important roles in osteosarcoma progression and this molecule will be a potential chemo- or radiotherapeutic molecular target for osteosarcoma therapy.

Expression of PIN genes in rice (Oryza sativa L.): tissue specificity and regulation by hormones.

Twelve genes of the PIN family in rice were analyzed for gene and protein structures and an evolutionary relationship with reported AtPINs in Arabidopsis. Four members of PIN1 (designated as OsPIN1a-d), one gene paired with AtPIN2 (OsPIN2), three members of PIN5 (OsPIN5a-c), one gene paired with AtPIN8 (OsPIN8), and three monocot-specific PINs (OsPIN9, OsPIN10a, and b) were identified from the phylogenetic analysis. Tissue-specific expression patterns of nine PIN genes among them were investigated using RT-PCR and GUS reporter. The wide variations in the expression domain in different tissues of the PIN genes were observed. In general, PIN genes are up-regulated by exogenous auxin, while different responses of different PIN genes to other hormones were found.