Study on the correlation and difference of qualitative information among three types of UPLC-HRMS and potential generalization in metabolites annotation.

The variation of qualitative information among different types of mainstream hyphenated instruments of ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) makes data sharing and standardization, and further comparison of results consistency in metabolite annotation not easy to attain. In this work, a quantitative study of correlation and difference was first achieved to systematically investigate the variation of retention time (tR), precursor ion (MS1), and product fragment ions (MS2) generated by three typical UPLC-HRMS instruments commonly used in metabolomics area. In terms of the findings of systematic and correlated variation of tR, MS1, and MS2 between different instruments, a computational strategy for integrated metabolite annotation was proposed to reduce the influence of differential ions, which made full use of the characteristic (common) and non-common fragments for scoring assessment. The regular variations of MS2 among three instruments under four collision energy voltages of high, medium, low, and hybrid levels were respectively inspected with three technical replicates at each level. These discoveries could improve general metabolite annotation with a known database and similarity comparison. It should provide the potential for metabolite annotation to generalize qualitative information obtained under different experimental conditions or using instruments from various manufacturers, which is still a big headache in untargeted metabolomics. The mixture of standard compounds and serum samples with the addition of standards were applied to demonstrate the principle and performance of the proposed method. The results showed that it could be an optional strategy for general use in HRMS-based metabolomics to offset the difference in metabolite annotation. It has some potential in untargeted metabolomics.

Method comparison of sample pretreatment and discovery of differential compositions of natural flavors and fragrances for quality analysis by using chemometric tools.

Natural flavors and fragrances or their extracts have been widely used in a large variety of areas, including food, cosmetic, and tobacco industrial processes, among others. The compositions and intrinsic attributes of flavors and fragrances were related to many factors, such as species, geographical origin, planting environment, storage condition, processing method, and so on. This not only increased the difficulty in analyzing the product quality of flavors and fragrances, but also challenged the idea of "quality-by-design (QbD)". This work proposed an integrated strategy for precise discovery of differential compounds among different classes and subsequent quality analysis of complex samples through flavors and fragrances used in tobacco industry as examples. Three pretreatment methods were first inspected to effectively characterize the sample compositions, including direct injection (DI), thermal desorption (TD), and stir bar sorptive extraction (SBSE)-TD, coupled with gas chromatography-mass spectrometry (GC-MS) analysis to obtain characteristic information of samples of flavors and fragrances. Then, principal component analysis (PCA) was applied to discover the relation and difference between chromatographic fingerprints and peak table data once significant components were recognized in a holistic manner. Model population analysis (MPA) was then used to quantitatively extract the characteristic chemicals representing the quality differences among different classes of samples. Some differential marker compounds were discovered for difference analysis, including benzyl alcohol, latin acid, l-menthol acid, decanoic acid ethyl ester, vanillin, trans-o-coumaric acid, benzyl benzoate, and so on. Furthermore, partial least squares-discriminant analysis (PLS-DA) and support vector machine (SVM) were respectively applied to construct multivariate models for evaluation of quality differences and variations. It was found that the accuracy attains to 100% for sample classification. With the help of optimal sample pretreatment technique and chemometric methods, the strategy for quality analysis and difference discovery proposed in this work can be widely delivered to more areas of complex plants with good interpretability and high accuracy.

HDAC8 functions in spindle assembly during mouse oocyte meiosis.

HDAC8 is a class I histone deacetylase that functions in a variety of biological processes through its non-histone substrates. However, its roles during oocyte meiosis remain elusive. Here, we document that HDAC8 localizes at spindle poles and positively participates in the regulation of microtubule organization and spindle assembly in mouse oocytes. Depletion of HDAC8 by siRNA-based gene silencing results in various spindle defects and chromosome misalignment during oocyte meiotic maturation, accompanied by impaired kinetochore-microtubule attachments. Consequently, a higher incidence of aneuploidy is generated in HDAC8-depleted MII eggs. In addition, inhibition of HDAC8 activity with its selective inhibitor PCI-34051 phenocopies the spindle/chromosome defects resulting from HDAC8 depletion by siRNA injection. Finally, we find that HDAC8 is required for the correct localization of ϕ-tubulin to spindle poles. Collectively, these data reveal that HDAC8 plays a significant role in regulating spindle assembly and thus ensuring the euploidy in mouse eggs.

Biochemical properties of C78SC96S rhFGF-2: a double point-mutated rhFGF-2 increases obviously its activity.

Fibroblast growth factor-2 (FGF-2) is a multifunctional polypeptide that affects many cellular functions and phenomena. The wild-type recombinant human fibroblast growth factor rhFGF-2(W) and the mutant C78SC96S rhFGF-2(M) were expressed in Escherichia coli and their products were purified. The results by the means of fluorescence spectroscopy and CD spectrums, suggested that due to its decreased hydrophobicity rhFGF-2 is not deposited as an inclusion body. The mitogenic activity of the expressed rhFGF-2(M) on 3T3 fibroblasts was shown to be 10-fold more than the expressed rhFGF-2(W) of which the biological activity was a little less than that of the standard rhbFGF(W), indicating that the increased biological activity was due to the change of its secondary structure, dimerization and affinity binding to FGF receptor (FGFR).