Spontaneous Mutations in HIV-1 Gag, Protease, RT p66 in the First Replication Cycle and How They Appear: Insights from an In Vitro Assay on Mutation Rates and Types.

While drug resistant mutations in HIV-1 are largely credited to its error prone HIV-1 RT, the time point in the infection cycle that these mutations can arise and if they appear spontaneously without selection pressures both remained enigmatic. Many HIV-1 RT mutational in vitro studies utilized reporter genes (LacZ) as a template to investigate these questions, thereby not accounting for the possible contribution of viral codon usage. To address this gap, we investigated HIV-1 RT mutation rates and biases on its own Gag, protease, and RT p66 genes in an in vitro selection pressure free system. We found rare clinical mutations with a general avoidance of crucial functional sites in the background mutations rates for Gag, protease, and RT p66 at 4.71 × 10-5, 6.03 × 10-5, and 7.09 × 10-5 mutations/bp, respectively. Gag and p66 genes showed a large number of 'A to G' mutations. Comparisons with silently mutated p66 sequences showed an increase in mutation rates (1.88 × 10-4 mutations/bp) and that 'A to G' mutations occurred in regions reminiscent of ADAR neighbor sequence preferences. Mutational free energies of the 'A to G' mutations revealed an avoidance of destabilizing effects, with the natural p66 gene codon usage providing barriers to disruptive amino acid changes. Our study demonstrates the importance of studying mutation emergence in HIV genes in a RT-PCR in vitro selection pressure free system to understand how fast drug resistance can emerge, providing transferable applications to how new viral diseases and drug resistances can emerge.

Effect of pre-moxibustion on apoptosis and proliferation of gastric mucosa cells.

AIM: To observe the effects of pre-moxibustion on apoptosis and proliferation of gastric mucosal cell in rats with stress-induced ulcer, and to analyze the relationship between those effects and the expression of heat-shock protein 70 (HSP70). METHODS: Sixty healthy Sprague Dawley rats were randomly assigned into four groups, namely group A, B, C and D. The animal model of stress ulcer was established by water immersion and restraint stress. The rats in group A, B, and D served as the restraint, model, and non-acupoint controls, respectively, while those in group C received moxibustion at Zusanli and Liangmen points. Immunohistochemical methodology was used to detect the expression of HSP70, apoptosis index (AI, multiply 10(-6)/microm(2)) and proliferation index (PCNA-LI, multiply 10(-6)/microm(2)). The mucosal expression of transforming growth factor alpha(TGF-alpha) was detected by radioimmunoassay. RESULTS: Moxibustion at Zusanli and Liangmen points significantly decreased the gastric injury and the apoptosis of gastric mucosal cells, while markedly increased the mucosal expression of TGF-alpha and HSP70 as well as the proliferation of gastric mucosal cells. Compared with group A, ulcer index (UI) (26.8 +/- 9.8 vs 12.0 +/- 5.9, P < 0.01), AI (9.6 +/- 4.2 vs 4.4 +/- 2.6, P < 0.05) and expression of HSP70 (9.6 +/- 4.2 vs 4.4 +/- 2.6, P < 0.05) were significantly increased, but the content of TGF-alpha (104.7 +/- 51.2 pg/mL vs 254.0 +/- 86.9 pg/mL, P < 0.01) and PCNA-LI (6.9 +/- 4.7 vs 14.9 +/- 4.6, P < 0.05) were significantly decreased in group B. However, ulcer index values (UI) and AI were obviously lower in group C compared to groups B and D (14.1 +/- 5.4 vs 26.8 +/- 9.8 and 26.2 +/- 7.7, P < 0.01; 3.0 +/- 1.6 vs 9.6 +/- 4.2 and 8.2 +/- 5.2, P < 0.05, respectively), but content of TGF-alpha (237.0 +/- 72.6 pg/mL vs 104.7 +/- 51.2 pg/mL and 154.1 +/- 61.3 pg/mL, P < 0.01) and expression of HSP70 (0.13 +/- 0.03 vs 0.08 +/- 0.06 and 0.06 +/- 0.04, P < 0.05) were higher in group C. Furthermore, the PCNA-LI was significantly higher in group C than in group B (21.6 +/- 4.1 vs 6.9 +/- 4.7, P < 0.01). CONCLUSION: Moxibustion at Zusanli and Liangmen points has a protective effect on rats gastric mucosa in stress-induced gastric ulcer, which is closely related to its actions in promoting synthesis of TGF-alpha and proliferation of gastric mucosal cells, suppressing gastric mucosal cell apoptosis, and up-regulating HSP70 expression.