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.

Integrated metabolism, network pharmacology, and pharmacokinetics to explore the exposure differences of the pharmacodynamic material basis in vivo caused by different extraction methods for Saussurea involucrata.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea involucrata Kar.et Kir. (S.I.) has long been used as a precious national medicine and clinically proven to be an effective treatment for rheumatoid arthritis (RA) and cardiovascular diseases. In clinical practice, two extraction methods of S.I., including water decoction and alcohol extraction, are prescribed to treat the same conditions. Nevertheless, no study has been performed on the exposure differences of the pharmacodynamic material basis in vivo caused by different extraction methods. AIM OF THE STUDY: Based on the integrated strategy of metabolism, network pharmacology, and pharmacokinetics, we aimed to reveal exposure differences in pharmacodynamic substances caused by different extraction methods. MATERIALS AND METHODS: Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) was employed to identify the chemical constituents of S.I. extracts and the metabolites in vivo after administration. Based on the analysis of prototype components in vivo, the major exposure active constituents, potential therapeutic targets and possible pharmacological mechanisms in RA treatment were investigated using network pharmacological analysis. Seven critical active components, including quercetin, hispidulin, apigenin, chlorogenic acid, arctigenin, syringin, and umbelliferone, were quantitatively compared between the alcohol, and aqueous extraction methods, which had been confirmed by the reference substance. RESULTS: The chemical comparison demonstrated that the types of chemicals in the two extracts were identical, mainly flavonoids, phenylpropanoids, coumarins, lignins, sesquiterpene lactones, and others, but the contents of the primary constituents in the aqueous extract were lower than those of the alcohol extract. A total of 30 prototype components and 174 metabolites were analyzed and identified in rat plasma, urine, fecal, and bile samples. Twenty-three prototype components were analyzed by network pharmacology, and seven critical active components were selected as representative markers for the pharmacokinetic study. Pharmacokinetic studies had shown that the Tmax values of apigenin, hispidulin, chlorogenic acid, arctigenin, and syringin after the oral administration of the alcohol extract were lower than those after the oral administration of the aqueous extract, and the above components in the alcohol extract could increase the absorption. Compared with the aqueous extract group, the Tmax and T1/2 of quercetin and umbelliferone were longer; it was suggested that alcohol extraction might have a slow-release and long-term effect on these two components. The relative bioavailability of apigenin, hispidulin, quercetin, chlorogenic acid, and umbelliferone in the alcohol extract group were higher than those in the aqueous extract group, which was consistent with the traditional clinical experience that alcohol extract could improve the efficacy of S.I. CONCLUSIONS: The major exposure active constituents in vivo were screened. The representative components that could be used in pharmacokinetics were determined by integrating network pharmacology and metabolism studies. The critical active compounds were quantitatively compared between the alcohol and aqueous extraction methods. This study clarified that flavonoids, coumarin, and phenylpropanoids might be the primary material basis that caused the exposure differences between aqueous and alcoholic extracts from S.I.. This research aimed to provide the basis of metabolism in vivo for further studying these pharmacodynamic differences.

Water-Soluble Cyclophanes Synthesized via the Zincke Reaction.

By taking advantage of the Zincke reaction, we successfully synthesized three macrocycles, each of which contains two bipyridinium units as the electron acceptors. Two of them contain sp2-hybridized atoms exclusively in the ring frameworks, while the third contains two methylene units. The third macrocycle is able to form 1:1 inclusion complexes with guests of complementary sizes. A pair of isomers, namely, phenanthrene and anthracene, could be separated by the third macrocycle.

[Seasonal variation of phytoplankton community structure in the Tianhe Reservoir of the Daluoshan Mountain in southern Zhejiang Province, China and its indication to water quality]. / 浙南大罗山天河水库浮游植物群落结构的季节变化及对水质的指示作用.

To explore the association between phytoplankton community structure and water quality of the Tianhe Reservoir in Wenzhou, Zhejiang, we examined the phytoplankton and physico-chemical factors in the Tianhe Reservoir from February 2019 to January 2020. A total of 89 phytoplankton species belonging to 60 genera and 7 phyla were identified. The annual average abundance and biomass of phytoplankton were 2.02×105 cells·L-1 and 0.26 mg·L-1 respectively. Bacillariophyta was dominant in the whole year. Chlorophyta was dominant in spring and autumn. Cyanophyta was dominant in summer. Both Euglenophyta and Pyrrophyta appeared in the whole year. Cryptophyta appeared in autumn and winter. However, none of them was dominant due to their low abundance. The dominant species throughout the year were Melosira granulata, Synedra acus, Navicula cryptocephala, Asterionella formosa, Pseudoanabaena, Closterlum venus, Pediastrum duplex, Peridinium bipes, Dinobryon cylindricum and Cryptomonas ovata. Results of redundancy analysis (RDA) showed that water temperature, total nitrogen, CODMn and pH value were the main factors driving phytoplankton community structure in the whole year. According to the evaluation based on diversity indices, including Shannon index, Pielou evenness index, Margalef richness index and comprehensive trophic status index (TLI), water quality of Tianhe Reservoir is between oligotrophication and mesotrophication.

Construction and evaluation of red blood cells-based drug delivery system for chemo-photothermal therapy.

In this study, a novel tumor-targeting drug delivery system (DDS) based on red blood cells (RBCs) were fabricated for combinational chemo-phototherapy against cancer. Cyclic peptide (cRGD) and indocyanine green (ICG) were applied to the surface of RBCs to increase the targeting and photothermal effect, respectively. Doxorubicin (DOX) as a model drug was loaded into RBCs by the hypotonic dialysis method. A series of tests have been carried out to evaluate the RBCs-based DDS and these tasks include physicochemical properties, cellular uptake, targeting ability, and combination therapeutic efficiency. As a result, the DOX was successfully loaded into RBCs and the drug loading amount was 0.84 ±â€¯0.09 mg/mL. There was no significant change of particle size after surface modification of RBCs. The RBCs-based DDS could target to the surface of cancer cells, which delivery DOX to the lesions efficiently and accurately. Meanwhile, due to the combined treatment effect, the RBCs-based DDS can effectively inhibit tumor growth. The RBCs-based DDS constructed in this research may have promising applications in cancer therapy due to their highly synergistic efficient therapy and to investigate its possibility for tumor therapy.

Mechanism-Driven Metabolic Engineering for Bio-Based Production of Free R-Lipoic Acid in Saccharomyces cerevisiae Mitochondria.

Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure R-lipoic acid, which has superior bioactivity than the S form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of S-lipoic acid or precursors with undesired chirality. Toward sustainable production of R-lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast Saccharomyces cerevisiae to facilitate metabolic engineering of the microbe for producing free R-lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized in vitro the activity of a lipoamidase from Enterococcus faecalis for releasing free R-lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled de novo free R-lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free R-lipoic acid biosynthesis in S. cerevisiae. We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable R-lipoic acid production.

Integration of Single Oriented Oxide Superlattices on Silicon Using Various Template Techniques.

To benefit from the diverse functionalities of perovskite oxides in silicon-based complementary metal oxide semiconductor (CMOS) technology, integrating oxides into a silicon platform has become one of the major tasks for oxide research. Using the deposition of LaMnO3/SrTiO3 (STO) superlattices (SLs) as a case study, we demonstrate that (001) single oriented oxide SLs can be integrated on Si using various template techniques, including a single-layer buffer of STO prepared by molecular beam epitaxy (MBE) and pulsed laser deposition, a multilayer buffer of Y-stabilized zirconia/CeO2/LaNiO3/STO, and STO-coated two-dimensional nanosheets of Ca2Nb3O10 (CNO) and reduced graphene oxide. The textured SL grown on STO-coated CNO nanosheets shows the highest crystallinity, owing to the small lattice mismatch between CNO and STO as well as less clamping from a Si substrate. The epitaxial SL grown on STO buffer prepared by MBE suffers the largest thermal strain, giving rise to a strongly suppressed saturation magnetization but an enhanced coercive field, as compared to the reference SL grown on an STO single crystal. These optional template techniques used for integrating oxides on Si are of significance to fulfill practical applications of oxide films in different fields.

Grape seed proanthocyanidins protect against streptozotocin­induced diabetic nephropathy by attenuating endoplasmic reticulum stress­induced apoptosis.

Diabetic nephropathy (DN) is by far the most common cause of end­stage renal disease (ESRD) in industrial countries, accounting for ~45% of all new ESRD cases in the United States. Grape seed proanthocyanidin extracts (GSPE) are powerful antioxidants, with an antioxidant ability 50­fold greater than that of vitamin E and 20­fold greater than that of vitamin C. The present study investigated whether GSPE can protect against streptozotocin (STZ)­induced DN and aimed to elucidate a possible mechanism. Male Sprague Dawley rats were randomly divided into three groups: Control group (N), diabetes mellitus group (DM) injected with 40 mg/kg STZ, and the GSPE treatment group (intragastric administration of 250 mg/kg/day GSPE for 16 weeks after diabetes was induced in the rats). Blood and kidney samples were collected after treatment. The renal pathological changes were determined with periodic acid­Schiff (PAS) staining, while the protein expression levels of glucose­regulated protein 78 (GRP78), phosphorylated­extracellular signal­regulated kinase (p­ERK) and Caspase­12 were determined by western blotting and immunohistochemical staining. Apoptosis was determined with a terminal deoxynucleotidyl transferase dUTP nick­end labeling (TUNEL) assay. Compared with the DM group, the GSPE group had no significant changes in the blood urea nitrogen (BUN) level and serum creatinine (Scr) level, but showed a significant decline in the renal index (RI) level and 24­h urinary albumin level (P<0.05). The histopathology results indicated very little pathological damage in the GSPE group. Compared with the DM group, the GSPE group had a significantly reduced number of TUNEL­positive cells (P<0.05), and the GSPE group had an obvious reduction in the protein expression of GRP78, p­ERK, and Caspase­12 (P<0.05). In this study, the results indicated that GSPE can protect renal function and attenuate endoplasmic reticulum stress­induced apoptosis via the Caspase­12 pathway in STZ­induced DN.

Improved Stable Indocyanine Green (ICG)-Mediated Cancer Optotheranostics with Naturalized Hepatitis B Core Particles.

In recent years, hepatitis B core protein virus-like particle (HBc VLP) is an impressive biomaterial, which has attracted considerable attention due to favorable properties such as structural stability, high uptake efficiency, and biocompatibility in biomedical applications. Heretofore, only a few attempts have been made to apply it in physical, chemical, and biological therapy for cancer. In this study, a tumor-targeting RGD-HBc VLP is first fabricated through genetic engineering. For image-guided cancer phototherapy, indocyanine green (ICG) is loaded into RGD-HBc VLP via a disassembly/reassembly pathway and electrostatic attraction with high efficiency. The self-assembled stable RGD-HBc VLP significantly improves body retention (fourfold longer), aqueous stability, and target specificity of ICG. Remarkably, these positive reformations promote more accurate and sensitive imaging of U87MG tumor, as well as prolonged tumor destruction in comparison with free ICG. Moreover, the photothermal and photodynamic effect on tumors are quantitatively differentiated by multiple linear regression analysis. Overall, less-potent medicinal ICG can be perfectly rescued by bioengineered HBc VLP to realize enhanced cancer optotheranostics.

A network meta-analysis in comparing prophylactic treatments of radiotherapy-induced oral mucositis for patients with head and neck cancers receiving radiotherapy.

OBJECTIVES: The objective of this network meta-analysis is to optimize the prophylactic treatment for radiotherapy-induced oral mucositis in patients with head and neck cancer (HNSCC) receiving postoperative or definitive radiotherapy with or without chemotherapy. MATERIALS AND METHODS: We searched electronic databases to identify all eligible randomized clinical trials on oral mucositis. The endpoint was grade 0-2 oral mucositis. Odds ratios (OR) and the corresponding 95% confidence intervals (CI) were extracted. Network meta-analysis was performed using the frequentist approach to conduct multiple treatment comparisons. RESULTS: In total, 57 trials with 5261 patients were eligible for this study. Both direct and network meta-analysis revealed that low-level laser additional to standard oral care (SOC) was better than most of the other treatments and achieved the highest effect on grade 0-2 oral mucositis, with a surface under the cumulative ranking curve (SUCRA) of 95.8%; however, SOC with or without placebo had worse effect than most of the other treatments and was ranked worst (SUCRA = 0.4%). Moreover, sensitivity analysis performed after we had combined the SOC and placebo groups (non-medication treatment, NMT) yielded similar results, with SUCRA of 91.3% and 3.5% for low-level laser and NMT, respectively. CONCLUSIONS: Low-level laser additional to SOC may be a more effective prophylactic treatment for reducing severe radiotherapy-induced oral mucositis; SOC alone is insufficient for patients with HNSCC receiving postoperative or definitive radiotherapy with or without chemotherapy.