Mechanism of zinc ejection by disulfiram in nonstructural protein 5A.

Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.

Application of hemoperfusion in severe acute organophosphorus pesticide poisoning

Background/aim: The aim of this research is to investigate the clinical efficacy of hemoperfusion in the treatment of severe acute organophosphorus pesticide poisoning (AOPP). Materials and methods: Patients meeting the inclusion criteria were divided into Groups 1 and 2 according to whether hemoperfusion was applied or not. Group 2 was observed as the control group. Conventional therapy for AOPP was given to Groups 1 and 2. Besides conventional treatment, patients in Group 1 were also treated with hemoperfusion therapy. Cholinesterase activity and blood glucose concentration were tested before hemoperfusion and for the first 3 days afterwards. The recovery time of 50% cholinesterase was recorded. At the same time, the incidence and mortality of intermediate syndrome was observed and compared. Results: The incidence and mortality of intermediate syndrome in Group 1 was obviously decreased, and the recovery time of cholinesterase activity was significantly shortened compared with Group 2. Conclusion: Hemoperfusion, used for treating severe AOPP, contributes to the improvement of cholinesterase activity, low incidence and mortality of intermediate syndrome, and increase in curative rate.

Effects of hemodialysis combined with hemoperfusion on severe acute pancreatitis.

BACKGROUND/AIMS: Severe acute pancreatitis (SAP) is characterized by persistent organ failure. This research aimed to evaluate the effect of hemodialysis combined with hemoperfusion on SAP. MATERIALS AND METHODS: Thirty-seven patients who were treated with hemoperfusion combined with hemodialysis were included in group O, and 31 patients treated with conventional therapy and hemoperfusion were included as control (group C). Leukocyte count, neutrophil percentage, amylase (AMY), blood urine nitrogen (BUN), creatinine (Cr), and total bilirubin (TBIL) were noted. The time when symptoms disappeared as well as complications after treatment was recorded. RESULTS: Leukocyte count, neutrophil percentage, AMY, BUN, Cr, and TBIL in two groups were remarkably decreased after treatment. However, these indexes were significantly lower in group O than those in group C after treatment, especially the neutrophil percentage, AMY, BUN, Cr, and TBIL. The time when the symptoms disappeared was 3.01±1.02 days in group O, which was shorter than 5.56±1.88 days in group C. There were 4 patients with acute renal failure and 2 patients had multiple organ failure in group C after treatment. But only 1 patient developed acute renal failure in group O. The difference in complications between two groups was significant (p<0.024). CONCLUSION: The combination of hemodialysis and hemoperfusion could have a better effect on SAP in removing toxic metabolites and inflammation mediators. It not only shortens the time of symptoms disappearing but also decreases the incidence of complications and the mortality.

Associations of MMP-2 and MMP-9 gene polymorphism with ulinastatin efficacy in patients with severe acute pancreatitis.

We aim to explore the associations between matrix metalloproteinase (MMP) MMP-2/MMP-9 gene polymorphism with ulinastatin (UTI) efficacy in treating severe acute pancreatitis (SAP). A total of 276 SAP patients were assigned into the control (n=135) and observation (n=141) groups. PCR-restriction fragment length polymorphism (PCR-RFLP) was used for genotype and allele frequency distribution. Relevance of MMP-2/MMP-9 genotypes with UTI efficacy was analyzed. The observation group showed lowered duration in symptoms (abdominal distension, abdominal pain, tenderness, and rebound tenderness) than the control group. Laboratory analysis (serum calcium, white blood cells, serum amylase, urine amylase, APACHE-II, and Balthazar CTIS scores) were decreased, while serum albumin levels increased after 7th day of therapy. The total effective rate of UTI for patients with MMP-2 C-1306T C/C genotype was higher than those with C/T and T/T genotypes after the 7th day of therapy, which was lower in patients with MMP-9 C-1562T C/C and C/T genotypes than those with T/T genotype. The duration for symptoms in patients with MMP-9 C-1562T T/T genotype was shorter than those with C/C and C/T genotypes, which was less in patients with MMP-2 C-1306T C/C genotype than those with C/T and T/T genotypes. The improvement values of APACHE-II and Balthazar CTIS scores for patients with MMP-2 C-1306T C/C genotype were higher than those with C/T and T/T genotypes, which for patients with MMP-9 C-1562T C/C and C/T genotypes were lower than those with T/T genotype. These results demonstrated that MMP-2/MMP-9 gene polymorphism was associated with UTI efficacy for SAP.

Spectrofluorimetric determination of trace nitrite with o-phenylenediamine enhanced by hydroxypropyl-ß-cyclodextrin.

A simple and sensitive spectrofluorimetric method was developed for the determination of nitrite in environmental and food samples. The method was based on the selective reaction of o-phenylenediamine with nitrite in acidic medium to form benzotriazole, which exhibited strong fluorescence at 568 nm with excitation at 420 nm in alkaline medium. The detection limit and sensitivity of the proposed method were improved by hydroxypropyl-ß-cyclodextrin through complexation. The linear calibration range for nitrite was 0.04-0.8 µg mL(-1) with a detection limit of 13.6 ng mL(-1) (S/N=3.29). The relative standard deviation for ten determinations of 0.1 and 0.4 µg mL(-1) nitrite were 1.38% and 2.01%, respectively. Twenty-eight coexistent ions were examined, and no serious interference for most of ions was observed. The proposed method was successfully applied for the determination of nitrite in the water, sausage and soil samples with recoveries of 95.5-108.5%. The results were in good agreement with the recommended AOAC method.