Non-canonical amino acids uncover the significant impact of Tyr671 on Taq DNA polymerase catalytic activity.

Responsible for synthesizing the complementary strand of the DNA template, DNA polymerase is a crucial enzyme in DNA replication, recombination and repair. A highly conserved tyrosine (Tyr), located at the C-terminus of the O-helix in family A DNA polymerases, plays a critical role in enzyme activity and fidelity. Here, we combined the technology of genetic code extension to incorporate non-canonical amino acids and molecular dynamics (MD) simulations to uncover the mechanisms by which Tyr671 impacts substrate binding and conformation transitions in a DNA polymerase from Thermus aquaticus. Five non-canonical amino acids, namely l-3,4-dihydroxyphenylalanine (l-DOPA), p-aminophenylalanine (pAF), p-acetylphenylalanine (pAcF), p-cyanophenylalanine (pCNF) and p-nitrophenylalanine (pNTF), were individually incorporated at position 671. Strikingly, Y671pAF and Y671DOPA were active, but with lower activity compared to Y671F and wild-type. Y671pAF showed a higher fidelity than the Y671F, despite both possessing lower fidelity than the wild-type. Metadynamics and long-timescale MD simulations were carried out to probe the role of mutations in affecting protein structure, including open conformation, open-to-closed conformation transition, closed conformation, and closed-to-open conformation transition. The MD simulations clearly revealed that the size of the 671 amino acid residue and interactions with substrate or nearby residues were critical for Tyr671 to determine enzyme activity and fidelity.

High-dose vitamin D(3) during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial.

BACKGROUND: Vitamin D was used to treat tuberculosis in the pre-antibiotic era, and its metabolites induce antimycobacterial immunity in vitro. Clinical trials investigating the effect of adjunctive vitamin D on sputum culture conversion are absent. METHODS: We undertook a multicentre randomised controlled trial of adjunctive vitamin D in adults with sputum smear-positive pulmonary tuberculosis in London, UK. 146 patients were allocated to receive 2·5 mg vitamin D(3) or placebo at baseline and 14, 28, and 42 days after starting standard tuberculosis treatment. The primary endpoint was time from initiation of antimicrobial treatment to sputum culture conversion. Patients were genotyped for TaqI and FokI polymorphisms of the vitamin D receptor, and interaction analyses were done to assess the influence of the vitamin D receptor genotype on response to vitamin D(3). This trial is registered with ClinicalTrials.gov number NCT00419068. FINDINGS: 126 patients were included in the primary efficacy analysis (62 assigned to intervention, 64 assigned to placebo). Median time to sputum culture conversion was 36·0 days in the intervention group and 43·5 days in the placebo group (adjusted hazard ratio 1·39, 95% CI 0·90-2·16; p=0.14). TaqI genotype modified the effect of vitamin D supplementation on time to sputum culture conversion (p(interaction)=0·03), with enhanced response seen only in patients with the tt genotype (8·09, 95% CI 1·36-48·01; p=0·02). FokI genotype did not modify the effect of vitamin D supplementation (p(interaction)=0·85). Mean serum 25-hydroxyvitamin D concentration at 56 days was 101·4 nmol/L in the intervention group and 22·8 nmol/L in the placebo group (95% CI for difference 68·6-88·2; p<0·0001). INTERPRETATION: Administration of four doses of 2·5 mg vitamin D(3) increased serum 25-hydroxyvitamin D concentrations in patients receiving intensive-phase treatment for pulmonary tuberculosis. Vitamin D did not significantly affect time to sputum culture conversion in the whole study population, but it did significantly hasten sputum culture conversion in participants with the tt genotype of the TaqI vitamin D receptor polymorphism. FUNDING: British Lung Foundation.

[Establishment and optimization of sliver staining differential display of microtubers from Pinellia ternata in vitro].

OBJECTIVE: In this study, orthogonal design was used to optinize DDRT-PCR amplification system on Pinellia ternata microtubers in vitro in five factors four levels respectively. METHOD: P. ternata stems and microtubers in vitro were selected as explants. The effects of five kinds of factors were studied by orthogonal design method including emplate cDNA, Mg2+, dNTPs, primers and Taq DNA polymerase, and in order to establish the optimum DDRT-PCR system of P. ternata microtubers in vitro. RESULT AND CONCLUSION: A satisfactory DDRT-PCR technique system for P. ternata microtubers in vitro with desirable repeatability and polymorphic bands was established. In a total volume of 20 microL DDRT-PCR system, it contained 10 x buffer, 150 micromol L(-1) dNTPs, 2 micromol L(-1) anchor primer, 1 micromol L(-1) arbitrary primer, 2.5 mmol L(-1) Mg2+, 0.6 U Taq DNA polymerase and 2.5 microg template cDNA. The effect of the five factors was in sequence of Taq DNA polymerase > template cDNA > dNTPs > Mg2+ > Primers. The optimum DDRT-PCR system will provide scientific reference basis for studying effecting character of P. ternata microtubers associated with genes expression.