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.

Dual role of Nrf2/HO-1 pathway in Z-ligustilide-induced ferroptosis against AML cells.

BACKGROUND: The scarcity of drugs targeting AML cells poses a significant challenge in AML management. Z-Ligustilide (Z-LIG), a phthalide compound, shows promising pharmacological potential as a candidate for AML therapy. However, its precise selective mechanism remains unclear. PURPOSE: In order to assess the selective inducement effects of Z-LIG on ferroptosis in AML cells and explore the possible involvement of the Nrf2/HO-1 pathway in the regulation of ferroptosis. METHODS: Through in vitro cell proliferation and in vivo tumor growth tests, the evaluation of Z-LIG's anticancer activity was conducted. Ferroptosis was determined by the measurement of ROS and lipid peroxide levels using flow cytometry, as well as the observation of mitochondrial morphology. To analyze the iron-related factors, western blot analysis was employed. The up-regulation of the Nrf2/HO-1 axis was confirmed through various experimental techniques, including CRISPR/Cas9 gene knockout, fluorescent probe staining, and flow cytometry. The efficacy of Z-LIG in inducing ferroptosis was further validated in a xenograft nude mouse model. RESULTS: Our study revealed that Z-LIG specifically triggered lipid peroxidation-driven cell death in AML cells. Z-LIG downregulated the total protein and nuclear entrance levels of IRP2, resulting in upregulation of FTH1 and downregulation of TFR1. Z-LIG significantly increased the susceptibility to ferroptosis by upregulating ACSL4 levels and simultaneously suppressing the activity of GPX4. Notably, the Nrf2/HO-1 pathway displayed a twofold impact in the ferroptosis induced by Z-LIG. Mild activation suppressed ferroptosis, while excessive activation promoted it, mainly driven by ROS-induced labile iron pool (LIP) accumulation in AML cells, which was not observed in normal human cells. Additionally, Nrf2 knockout and HO-1 knockdown reversed iron imbalance and mitochondrial damage induced by Z-LIG in HL-60 cells. Z-LIG effectively inhibited the growth of AML xenografts in mice, and Nrf2 knockout partially weakened its antitumor effect by inhibiting ferroptosis. CONCLUSION: Our study presents biological proof indicating that the selective initiation of ferroptosis in leukemia cells is credited to the excessive activation of the Nrf2/HO-1 pathway triggered by Z-LIG.

Effectiveness comparisons of acupuncture for psoriasis: A Bayesian network meta-analysis protocol.

BACKGROUND: Psoriasis is an immune-mediated polygenic hereditary skin disease quality of the patients' life because of the great trouble it causes to patients. Whereas there is variability when we regard the selection of acupuncture treatments in practice and most choices are made based on personal experience or preference of clinician. This study uses network meta-analysis to compare the effectiveness of different forms of acupuncture for psoriasis and assesses the evidence with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. METHODS: We will search for PubMed, Cochrane Library, AMED, EMbase, WorldSciNet; Nature, Science online and China Journal Full-text Database (CNKI), China Biomedical Literature CD-ROM Database (CBM), and related randomized controlled trials included in the China Resources Database. The time is limited from the construction of the library to April 2019. The quality of the included RCTs will be evaluated by the risk of bias (ROB) tool and the evidence will be evaluated by GRADE. STATA 13.0 and WinBUGS 1.4.3 through the GeMTC package will be used to perform a network meta-analysis to synthesize direct and indirect evidence. RESULTS: The results of this network meta-analysis (NMA) will be submitted to a peer-reviewed journal for publication. TRIAL REGISTRATION NUMBER: PROSPERO CRD42019123437.

Isolation and purification of immunoglobulin G from bovine colostrums by hydrophobic charge-induction chromatography.

Hydrophobic charge-induction chromatography, a novel chromatographic technique for bioseparation, was developed to isolate and purify bovine IgG with high purity. In this work, the raw IgG solution, a precipitate from bovine colostrum powder solution with 40% (wt/vol) ammonium sulfate, was dissolved in 50mM phosphate buffer and used as loading solution for investigating chromatographic conditions on a mercapto-ethyl-pyridine (MEP) HyperCel (Pall Corp., Port Washington, NY) sorbent. The initial IgG concentration had no effect on the dynamic binding capacity of MEP HyperCel resin, but the linear velocity of loading solution had an obvious effect on the dynamic IgG binding capacity and IgG recovery. The maximum linear velocity was optimized as 0.4cm/min of loading solution, and 90% recovery of IgG was achieved. Under these optimized binding conditions, the pH and ionic strength for the elution process were selected as pH 4.5 and 0.5 M NaCl, respectively. Subsequently, hydrophobic charge-induction chromatography was performed on a MEP HyperCel sorbent to isolate IgG using bovine colostrum whey as the loading solution. Under the optimized operation conditions, a remarkable process improvement in IgG purification was received, which includes a yield of 91.5%, a purity of 93.9% (wt/wt), and a purification factor of 6.8. The results indicated that MEP HyperCel chromatography offers an efficient means to purify IgG from bovine colostrums.

Characterization of a Baculovirus newly isolated from the tea slug moth, Iragoidae fasciata.

The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5Bl-4. Bioassays on lethal concentration (LC(50)) and lethal time (LT(50)) were conducted to test the susceptibility of I. fasciata larvae to the virus.

[Effects of Panax notoginseng saponins on antioxidation and preventing DNA damage caused by hydroxyl radical].

Scavening effects of panax notoginseng saponins on active oxygen species in the systems of Fenton reaction, autoxidation of pyrogallol and riboflavine photosensitization were investigated by the mean of spectrophotometry. The inhibition of panax notoginseg saponins on the damage of DNA chain induced by hydroxyl radical by panax notoginseng saponins was observed by the mean of gelose gel electrophoresi and thiobartital spectrophotometry. The results showed that panax notoginseng saponins could scaven * OH, O2*- efficiently and protect DNA chains from being damaged by hydroxyl radical.

[Taohong Siwu decoction II inhibits the angiogenesis and the expressions of VEGF and KDR/FLK-1 in C57BL/6J mice bearing B16 melanoma].

OBJECTIVE: To investigate the effects of Taohong Siwu decoction II on B16 melanoma in mice and the underlying mechanism. METHOD: C57BL/6J mice bearing B16 melanoma were used in this study. The experimental groups were treated respectively with Taohong Siwu decoction II at doses of 2.5, 5 and 10 g x kg(-1) and cyclophosphamide at 0.05 g x kg(-1), Taohong Siwu decoction II at 10 g x kg(-1) plus cyclophosphamide at 0.025 mg x kg(-1). The tumor volume and weight, the expression of VEGF and KDR/FLK-1 and the amount of MVD were measured. RESULT: The tumor volume and weight, the expression of VEGF and KDR/FLK-1 and the amount of MVD were reduced significantly after treatment with Taohong Siwu decoction II at the doses of 5 and 10 g x kg(-1) and Taohong Siwu decoction II combined with cyclophosphamide as compared with normal saline-treated group (P < 0.01). CONCLUSION: Taohong Siwu decoction II can inhibit the growth of B16 melanoma in mice and attenuate the expressions of VEGF and KDR/FLK-1, suggesting that Taohong Siwu decoction II produces the antitumor effect via a possible antiagiogenisis mechanism.