A series of computational methods for pK shift prediction are extensively tested on a set of benchmark protein systems, aiming at identifying pitfalls and evaluating their performance on high variants. Including 19 ASP residues in 10 protein systems, the benchmark set consists of both residues with highly shifted pK values as well as those varying little from the reference value, with an experimental RMS free energy differences of 2.49 kcal/mol with respect to blocked amino acid, namely the RMS pK shift being 1.82 pK units. The constant pH molecular dynamics (MD), alchemical methods, PROPKA3.1, and multiconformation continuum electrostatics give RMSDs of 1.52, 2.58, 1.37, and 3.52 pK units, respectively, on the benchmark set. The empirical scoring method is the most accurate one with extremely low computational cost, and the pH-dependent model is also able to provide accurate results, while the accuracy of MD sampling incorporating alchemical free energy simulation is prohibited by convergence achievement and the performance of conformational search incorporating multiconformation continuum electrostatics is bad. Former research works did not define statistical uncertainty with care and yielded the questionable conclusion that alchemical methods perform well in most benchmarks. In this work the traditional alchemical methods are thoroughly tested for high variants. We also performed the first application of nonequilibrium alchemical methods to the pK cases.
Defective DNA damage response is a threat to genome stability and a proven cause of tumorigenesis. C21ORF2 (chromosome 21 open reading frame 2) is a novel gene on chromosome 21, and the C21ORF2 protein is found to interact with NEK1. Earlier studies showed that C21ORF2 might be associated with some human genetic diseases including Down syndrome. However, the cellular functions of C21ORF2 remain unknown. In the present study, we reported that C21ORF2 affected cell proliferation after DNA damage induced by ionizing radiation, and DNA repair was less efficient in C21ORF2-depleted cells compared with control cells. However, C21ORF2-knockdown cells did not show defects in the activation of the G2-phase DNA damage checkpoint. Furthermore, homologous recombination, but not non-homologous end joining repair, was found to be impaired after C21ORF2 attenuation, which could be rescued by the overexpression of NEK1, indicating that C21ORF2 functions in the same pathway as NEK1 in DNA damage repair.
The dissipation behaviour of three neonicotinoids - thiamethoxam, imidacloprid and acetamiprid - was compared in tea shoots, in Chinese green and black tea, and after tea infusion in hot water. The simple and rapid analytical procedures for the quantification of these three residues in these matrices were developed using HPLC with ultraviolet (UV) detection. Degradation rates in tea shoots of neonicotinoids applied in either recommended or double dosages followed first-order kinetics, with half-lives of 1.62 or 1.58 days for thiamethoxam, of 2.45 or 2.67 days for imidacloprid, and of 3.24 or 3.85 days for acetamiprid, respectively. Through harvest and processing the residue retentions for thiamethoxam, imidacloprid and acetamiprid were 85.0%, 84.1% and 70.6% of the initial dosages in green tea, and 77.1%, 52.4% and 57.4% in black tea. These three residues all showed high transfer rates through green or black tea brewing of 80.5% or 81.6% for thiamethoxam, of 63.1% or 62.2% for imidacloprid, and of 78.3% or 80.6% for acetamiprid. Waiting periods between the last application and harvest of at least 12, 17 and 20 days were suggested for thiamethoxam, imidacloprid and acetamiprid, respectively, after application at their recommend dosages to ensure levels below a maximum residue limit (MRL) of 0.05 mg kg(-1).
Tea leaves contain an extraordinarily high level of flavonoids that contribute to tea health benefits and flavor characteristics, but the regulatory mechanism of ambient ultraviolet B (UV-B) on tea flavonoid enrichment remains unclear. Here, we report that ambient UV-B modulates tea quality by inducing a metabolic flux in flavonoid biosynthesis. UV-B absence decreased bitter- and astringent-tasting flavonol glycosides (kaempferol-7-O-glucoside, myricetin-3-O-glucoside, and quercetin-7-O-glucoside) but increased non-galloylated catechins. Conversely, supplementary UV-B increased flavonols and decreased catechins in tea leaves. These responses were achieved via CsHY5, which mediates the UV-B-induced MYB12 activation and binds to the promoters of flavonoid biosynthetic genes (CsFLS, CsLARa, and CsDFRa), leading to flavonoid changes. Transcriptomic data indicated that UV-B-induced tea flavonoid regulation is responsive to multiple biotic and abiotic environmental stresses. These findings improve our understanding of light-regulated tea astringency and bitterness underlying shading effects and seasonal light changes and provide novel insights into tea cultivation management and processing.
Sample preparation using an absorbent for removal of polyphenols and a solid-phase extraction (SPE) cartridge for cleanup followed by high-performance liquid chromatography (HPLC) has been investigated for the simultaneous determination of eight neonicotinoid insecticides (dinotefuran, nitenpyram, thiamethoxam, imidacloprid, clothianidin, imidaclothiz, acetamiprid, and thiacloprid). After tea samples were soaked with water and extracted with acetonitrile, sample extracts were treated with an appropriate amount of polyvinylpolypyrrolidone (PVPP) to effectively remove polyphenols. The treated extract was cleaned up with a Carb-PSA cartridge. Neonicotinoid insecticides were eluted with acetonitrile from the cartridge and dried. The extract was redissolved with methanol/water (1:9, v/v) and analyzed by conventional HPLC coupled with an ultraviolet detector. The recoveries of eight neonicotinoid insecticides in tea samples were 71.4-106.6% at 0.1-1.0 mg kg(-1) spiked levels. Relative standard deviations were <10% for all of the recovery tests. The established method was simple, effective, and accurate and could be used for monitoring neonicotinoid insecticides in tea.
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