Insights into resistance mechanism of hepatitis C virus nonstructural 3/4A protease mutant to boceprevir using umbrella sampling simulation study

Abstract

Hepatitis C virus can cause inflammation in human liver cells, leading to liver cirrhosis and liver cancer. Based on the World Health Organization reports, about 228 million people in the world have hepatitis C. To date, some inhibitory medicines against the hepatitis C virus nonstructural 3/4A protease, such as boceprevir, have entered clinical trial phases. However, several hepatitis C virus nonstructural 3/4A protease mutations have been recognized to decrease susceptibility of boceprevir to hepatitis C virus. The molecular details behind inhibitor resistance of these single-point mutations are not still understood. Thus, in this research, computational strategies were applied to clarify the inhibitor resistance mechanism. From umbrella sampling simulation and energy profiles, the polar interactions are the main driving force for boceprevir binding. Based on the analyzed R155T mutant, the main reason for the occurrence of boceprevir resistance is the conformation alterations of S4 and extended S2 binding pockets. These changes, lead to decreased binding ability of the key residues to P2 and P4 moieties of boceprevir. Moreover, structural results show that the disappearance of important salt bridges can bring about the great conformation changes of the binding pockets in R155T.

Communicated by Ramaswamy H. Sarma     

Oleanolic acid increases the anticancer potency of doxorubicin in pancreatic cancer cells

Combination therapy is a novel cancer therapy approach that combines two or more chemotherapy drugs. This treatment modality enhances the efficacy of chemotherapy by targeting key pathways in an additive or synergistic manner. Therefore, we investigated the efficacy of combination therapy by widely used chemotherapy drug doxorubicin (DOX) and oleanolic acid (OA) to induction of apoptosis for pancreatic cancer (PC) therapy. The effects of DOX, OA, and their combination (DOX-OA) were investigated on proliferation and viability of PC cell line (PANC-1) by MTT assay. Moreover, migration and invasion of the cancer cells were evaluated by trans-well migration assay and wound healing assay. Flow cytometry and DAPI (4′,6-diamidino-2-phenylindole) staining were employed to investigate apoptosis quantification and qualification of the treated cancer cells. Finally, mRNA expression of apoptosis-related genes was assessed by quantitative real-time polymerase chain reaction. Our results demonstrated that the proliferation and metastasis potential of PC cells significantly decreased after treatment by DOX, OA, and DOX-OA. Moreover, we observed an increase in apoptosis percentage in the treated cancer cells. The apoptosis-related gene expression was modified to increase the apoptosis rate in all of the treatment groups. However, the anticancer potency of DOX-OA combination was significantly more than that of DOX and OA treatments alone. Our study suggested that DOX-OA combination exerts more profound anticancer effects against PC cell lines than DOX or OA monotherapy. This approach may increase the efficiency of chemotherapy and reduce unintended side effects by lowering the prescribed dose of DOX.     

Non-antisense cellular responses to oligonucleotides

Oligonucleotides induce various cellular responses which are not due to the blockade of protein synthesis by an antisense mechanism. Oligonucleotides presenting double-stranded or G-quartet structures (ribo- or deoxyribonucleotides, phosphodiester or phosphorothioated) induce retraction of neurites and aggregation of chicken retinal cells within 10–20 h. This effect is reversible, non-toxic; it appears to require internalization and can be mimicked by treatment of the cells with an RGDS peptide. The oligonucleotides appear to trigger a cascade of intracellular events, affecting the adhesive properties of integrins. In addition, a subset of oligonucleotides induced platelet aggregation, probably through their interaction with membrane receptors. Recognition of these effects is important for the design and interpretation of antisense experiments.     

Flexibility of the shoulder joint measured as range of abduction in a large representative sample of men and women over 65 years of age

In a representative survey of 1000 elderly men and women aged over 65 years living in their own homes, assessments have been made of flexibility measured as range of shoulder abduction in addition to health status, psychological well-being and reported customary activity. The results for shoulder abduction were approximately normally distributed and the mean values (+/- 1 standard deviation) were as follows: - in men aged 65-74 years, 129 (+/- 14) degrees and aged over 74 years, 121 (+/- 19) degrees; in women aged 65-74 years, 124 (+/- 19) degrees and aged over 74 years, 114 (+/- 22) degrees. These mean values are about 30 degrees less than those accepted for younger subjects. Nearly half the distribution falls below the accepted threshold level of 120 degrees for adequate function. There were significant effects of sex and age (P less than 0.001); women had poorer flexibility and the reduction with age amounted to 10 degrees per decade. Multiple regression analysis showed that the effect of age was accounted for in part by health, strength and customary use. The effects of use were most marked in those with some disability. This suggests that maintained or increased use could offset some of the age-related loss of the range of shoulder movement.     

Effect of Piriformospora indica and Sebacina vermifera on plant growth and essential oil yield in Thymus vulgaris in vitro and in vivo experiments

Thymus vulgaris of family Lamiaceae is one of the most plants in pharmacy industries. In this study effect of Piriformospora indica and Sebacina vermifera on growth and development plant, yield and composition of the essential oil in a completely randomized design were evaluated in vitro and in pot culture experiments. Plants were studied by means of plant height, shoot fresh and dry weights, number of shoots, root length, root fresh and dry weights and essential oil analyses. The oil was extracted from the dry matter of shoots by hydro distillation, and their composition was determined by GC/MS. In vitro and in vivo cultures showed that plant height and root length increased in pots inoculated with S. vermifera and P. indica. Maximum fresh and dry weight (shoot and root), number of shoots were observed in pots inoculated with P. indica. In thyme inoculated with S. vermifera and P. indica oil yield increased as compared to non-inoculated control plants. GC and GC/MS revealed that the level of thymol was enhanced as the effect of S. vermifera and P. indica.     

Diazinon-induced ulcerative keratitis in C57bl/6 mice

As a well-known organophosphate insecticide, diazinon (DZN) has been used for several decades in agriculture. The major signs of ophthalmic toxicity of DZN have been reported to be cholinergic overstimulation (lacrimation, myosis). Here, we report, for the first time, ulcerative keratitis in C57bl/6 mice secondary to sub-acute exposure to DZN. Four groups of female C57bl/6 mice were administered intraperitoneally either DZN (1, 5, 25 mg/kg/day) or vehicle for 14 consecutive days. Then, histopathological examinations on mice eyes were performed using light microscope and scored for corneal keratitis. Furthermore, blood cholinesterase activity, and hematologic examinations were performed. Data indicated a significant ulcerative keratitis with prompt vision loss in mice exposed to 25 and 5 mg/kg/day (P < 0.05) doses. These results suggest that diazinon might induce ulcerative keratitis secondary to its immunosuppresive effects at high doses in C57bl/6 mice.