Detecting mitochondrial mutations associated with aminoglycoside ototoxicity by noninvasive prenatal testing.

OBJECTIVES: Numerous diseases and disorders are associated with mitochondrial DNA (mtDNA) mutations, among which m.1555A > G and m.1494C > T mutations in the 12 S ribosomal RNA gene contribute to aminoglycoside-induced and nonsyndromic hearing loss worldwide. METHODS: A total of 76,842 qualified non-invasive prenatal (NIPT) samples were subjected to mtDNA mutation and haplogroup analysis. RESULTS: We detected 181 m.1555A > G and m.1494C > T mutations, 151 of which were subsequently sequenced for full-length mitochondrial genome verification. The positive predictive values for the m.1555A > G and m.1494C > T mutations were 90.78% and 90.00%, respectively, a performance comparable to that attained with newborn hearing screening. Furthermore, mitochondrial haplogroup analysis revealed that the 12 S rRNA 1555A > G mutation was enriched in sub-haplotype D5[p = 0, OR = 4.6706(2.81-7.78)]. CONCLUSIONS: Our findings indicate that the non-invasive prenatal testing of cell-free DNA obtained from maternal plasma can successfully detect m.1555A > G and m.1494C > T mutations.

A retrospective analysis of preemptive pharmacogenomic testing in 22,918 individuals from China.

BACKGROUND: Pharmacogenomics (PGx) examines the influence of genetic variation on drug responses. With more and more Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines published, PGx is gradually shifting from the reactive testing of single gene toward the preemptive testing of multiple genes. But the profile of PGx genes, especially for the intra-country diversity, is not well understood in China. METHODS: We retrospectively collected preemptive PGx testing data of 22,918 participants from 20 provinces of China, analyzed frequencies of alleles, genotypes and phenotypes of pharmacogenes, predicted drug responses for each participant, and performed comparisons between different provinces. RESULTS AND CONCLUSION: After analyzing 15 pharmacogenes from CPIC guidelines of 31 drugs, we found that 99.97% of individuals may have an atypical response to at least one drug; the participants carry actionable genotypes leading to atypical dosage recommendation for a median of eight drugs. Over 99% of the participants were recommended a decreased warfarin dose based on genetic factors. There were 20 drugs with high-risk ratios from 0.18% to 58.25%, in which clopidogrel showed the highest high-risk ratio. In addition, the high-risk ratio of rasburicase in GUANGDONG (risk ratio (RR) = 13.17, 95%CI:4.06-33.22, p < 0.001) and GUANGXI (RR = 23.44, 95%CI:8.83-52.85, p < 0.001) were significantly higher than that in all provinces. Furthermore, the diversity we observed among 20 provinces suggests that preemptive PGx testing in different geographical regions in China may need to pay more attention to specific genes. These results emphasize the importance of preemptive PGx testing and provide essential evidence for promoting clinical implementation in China.

Binding Mechanism of CD47 with SIRPα Variants and Its Antibody: Elucidated by Molecular Dynamics Simulations.

The intricate complex system of the differentiation 47 (CD47) and the signal-regulatory protein alpha (SIRPα) cluster is a crucial target for cancer immunotherapy. Although the conformational state of the CD47-SIRPα complex has been revealed through crystallographic studies, further characterization is needed to fully understand the binding mechanism and to identify the hot spot residues involved. In this study, molecular dynamics (MD) simulations were carried out for the complexes of CD47 with two SIRPα variants (SIRPαv1, SIRPαv2) and the commercially available anti-CD47 monoclonal antibody (B6H12.2). The calculated binding free energy of CD47-B6H12.2 is lower than that of CD47-SIRPαv1 and CD47-SIRPαv2 in all the three simulations, indicating that CD47-B6H12.2 has a higher binding affinity than the other two complexes. Moreover, the dynamical cross-correlation matrix reveals that the CD47 protein shows more correlated motions when it binds to B6H12.2. Significant effects were observed in the energy and structural analyses of the residues (Glu35, Tyr37, Leu101, Thr102, Arg103) in the C strand and FG region of CD47 when it binds to the SIRPα variants. The critical residues (Leu30, Val33, Gln52, Lys53, Thr67, Arg69, Arg95, and Lys96) were identified in SIRPαv1 and SIRPαv2, which surround the distinctive groove regions formed by the B2C, C'D, DE, and FG loops. Moreover, the crucial groove structures of the SIRPα variants shape into obvious druggable sites. The C'D loops on the binding interfaces undergo notable dynamical changes throughout the simulation. For B6H12.2, the residues Tyr32LC, His92LC, Arg96LC, Tyr32HC, Thr52HC, Ser53HC, Ala101HC, and Gly102HC in its initial half of the light and heavy chains exhibit obvious energetic and structural impacts upon binding with CD47. The elucidation of the binding mechanism of SIRPαv1, SIRPαv2, and B6H12.2 with CD47 could provide novel perspectives for the development of inhibitors targeting CD47-SIRPα.

Conformational plasticity of SpyCas9 induced by AcrIIA4 and AcrIIA2: Insights from molecular dynamics simulation.

CRISPR-Cas9 systems constitute bacterial adaptive immune systems that protect against phage infections. Bacteriophages encode anti-CRISPR proteins (Acrs) that mitigate the bacterial immune response. However, the structural basis for their inhibitory actions from a molecular perspective remains elusive. In this study, through microsecond atomistic molecular dynamics simulations, we demonstrated the remarkable flexibility of Streptococcus pyogenes Cas9 (SpyCas9) and its conformational adaptability during interactions with AcrIIA4 and AcrIIA2. Specifically, we demonstrated that the binding of AcrIIA4 and AcrIIA2 to SpyCas9 induces a conformational rearrangement that causes spatial separation between the nuclease and cleavage sites, thus making the endonuclease inactive. This separation disrupts the transmission of signals between the protospacer adjacent motif recognition and nuclease domains, thereby impeding the efficient processing of double-stranded DNA. The simulation also reveals that AcrIIA4 and AcrIIA2 cause different structural variations of SpyCas9. Our research illuminates the precise mechanisms underlying the suppression of SpyCas9 by AcrIIA4 and AcrIIA2, thus presenting new possibilities for controlling genome editing with higher accuracy.

Low-frequency conductivity of low wear high-entropy alloys.

High-entropy alloys (HEAs) provide new research avenues for alloy combinations in the periodic table, opening numerous possibilities in novel-alloy applications. However, their electrical characteristics have been relatively underexplored. The challenge in establishing an HEA electrical conductivity model lies in the changes in electronic characteristics caused by lattice distortion and complexity of nanostructures. Here we show a low-frequency electrical conductivity model for the Nb-Mo-Ta-W HEA system. The cocktail effect is found to explain trends in electrical-conductivity changes in HEAs, while the magnitude of the reduction is understood by the calculated plasma frequency, free electron density, and measured relaxation time by terahertz spectroscopy. As a result, the refractory HEA Nb15Mo35Ta15W35 thin film exhibits both high hardness and excellent conductivity. This combination of Nb15Mo35Ta15W35 makes it suitable for applications in atomic force microscopy probe coating, significantly improving their wear resistance and atomic-scale image resolution.

A new stilbene dimer from Vitis amurensis.

One new stilbene dimer named amurensin O (1), together with one known resveratrol trimer melapinol B (2), was isolated from Vitis amurensis, and their structures were identified on the basis of spectral analysis, especially 2D NMR spectral analysis.

[Discussion of the characteristic indexs in using three-dimensional fluorescence method to identify different crude oils].

The present paper investigates the influence of solvent purity on the test of three-dimensional fluorescence spectra, determines the process of its pre-purification, and studies the influence of the concentrations on the three-dimensional fluorescence characteristic indexes such as the characteristic peaks position, fluorescence intensity (F), and ratio (R) of fluorescence intensity when the phenomena of fluorescence quenching won't appear. The result shows that the concentration affects the value F and value R largely but its effect on the relative peak intensity (R(s)) is relatively small. Different crude oils has different value R(s). According to those characteristics, this paper presents that the value R(s) can be used as a new characteristic index for identification of crude oil. This conclusion was confirmed by testing many crude oils stably and reliably from different areas with their concentrations at 5 mg x L(-1). This method can be applied to identify most crude oils preliminary and further accurate analysis can be carried out together with other index.

Oxidation of catechin and rutin by pentaammineruthenium(III) complexes.

The reaction of catechin and rutin with Ru(NH(3))(5)L(3+) (L = N-methylpyrazinium (pzCH(3)(+)), pyrazine (pz), and isonicotinamide (isn)) complexes underwent a two-electron oxidation on the catechol ring (B ring) with the formation of quinone products. The kinetics of the oxidation, carried out at [H(+)] = 0.01-1.0 M and pH = 4.0-7.6, suggested that the reaction process involves the rate determining one-electron oxidation of the flavonoids in the form of H(2)X (k(0)), HX(-) (k(1)), and X(2-) (k(2)) by Ru(NH(3))(5)L(3+) complexes to form the corresponding semiquinone radicals, followed by the rapid scavenge of the radicals by the Ru(III) complexes. The specific rate constants (k(0), k(1), and k(2)) were measured and the results together with the application of the Marcus theory were used to estimate the self-exchange parameters for the one-electron couples of the flavonoids, H(2)X/H(2)X(+*), HX(-)/HX(*), and X(2-)/X(-*).

Engineering human Fhit, a diadenosine triphosphate hydrolase, into an efficient dinucleoside polyphosphate synthase.

The putative human tumor suppressor gene FHIT encodes Fhit, the fragile histidine triad protein. Fhit is thought to participate in a signal transduction pathway involving dinucleoside polyphosphates. Fhit catalyzes the Mg2+-dependent hydrolysis of P1-5'-O-adenosine-P3-5'-O-adenosine triphosphate (Ap3A) to AMP and MgADP. Mutation of His96 to glycine disables Fhit as a catalyst for the hydrolysis of phosphoanhydrides such as Ap3A. However, the mutated enzyme H96G-Fhit efficiently catalyzes the synthesis of phosphoanhydride bonds in reactions of nucleoside-5'-phosphimidazolides with nucleoside di- and triphosphates. H96G-Fhit can be employed in the synthesis of a wide range of dinucleoside tri- and tetraphosphates. We here describe the use of H96G-Fhit to catalyze the synthesis of Ap3A, Ap3C, Ap3G, Ap3T, Ap3U, Cp3U, Tp3U, dAp3U, Ap4A, Ap4U, and the fluorescent Ap4etheno-C.

The mechanism of action of the fragile histidine triad, Fhit: isolation of a covalent adenylyl enzyme and chemical rescue of H96G-Fhit.

The human fragile histidine triad protein Fhit catalyzes the Mg(2+)-dependent hydrolysis of P(1)-5'-O-adenosine-P(3)-5'-O-adenosine triphosphate, Ap(3)A, to AMP and ADP. The reaction is thought to follow a two-step mechanism, in which the complex of Ap(3)A and Mg(2+) reacts in the first step with His96 of the enzyme to form a covalent Fhit-AMP intermediate and release MgADP. In the second step, the intermediate Fhit-AMP undergoes hydrolysis to AMP and Fhit. The mechanism is inspired by the chain-fold similarities of Fhit to galactose-1-phosphate uridylyltransferase, which functions by an analogous mechanism, and the observation of overall retention in configuration at phosphorus in the action of Fhit (Abend, A., Garrison, P. N., Barnes, L. D., and Frey, P. A. (1999) Biochemistry 38, 3668-3676). Direct evidence in support of this mechanism is reported herein. Reaction of Fhit with [8,8'-(3)H]-Ap(3)A and denaturation of the enzyme in the steady state leads to protein-bound tritium corresponding to 11% of the active sites. Similar experiments with the poor substrate MgATP leads to 0.9% labeling. The mutated protein H96G-Fhit is completely inactive against MgAp(3)A. However, it is chemically rescued by free histidine. H96G-Fhit also catalyzes the hydrolysis of adenosine-5'-phosphoimidazolide, AMP-Im, and of adenosine-5'-phospho-N-methylimidazolide, AMP-N-MeIm. The hydrolyses of AMP-Im and of AMP-N-MeIm by H96G-Fhit are thought to represent chemical rescue of the covalent Fhit-AMP intermediate. Wild-type Fhit is also found to catalyze the hydrolyses of AMP-Im and of AMP-N-MeIm nearly as efficiently as the hydrolysis of MgAp(3)A. The results indicate that Mg(2+) in the reaction of Ap(3)A is required for the first step, the formation of the covalent intermediate Fhit-AMP, and not for the hydrolysis of the intermediate in the second step.

An isorhapontigenin tetramer and a novel stilbene dimer from Gnetum hainanense.

The first isorhapontigenin tetramer, gnetuhainin R (1) and a novel dimeric stilbene, gnetuhainin S (2) were isolated from the lianas of Gnetum hainanense. Their structures were elucidated on the basis of spectroscopic and chemical evidence, especially 2D NMR analysis. The structure of 2 was further supported by X-ray crystallographic analysis. The anti-inflammatory activity of 1 has been tested, and it showed potent activity of histamine receptor antagonism.