In this study, melanin (BgM) was obtained from Boletus griseus. The chemical composition and structure of BgM were characterized by UV-visible absorption spectrum, Fourier transform infrared spectrum, elemental analysis, nuclear magnetic resonance, pyrolysis gas chromatography mass spectrometry, and ultra-performance liquid chromatography–high resolution mass spectrometry. The percentage contents of C, H, N, S and O elements were 56.38%, 5.86%, 6.17%, 2.44%, and 28.04%, and the S/N and C/N ratios were 0.17 and 10.66, respectively. The UV-vis spectrum of BgM showed a maximum absorption peak at 214 nm. Characteristic absorption peaks were observed at 3426, 1600 and 1105 cm−1, and BgM contained phenolic hydroxyl, amidogen, carbonyl, methylene, and methyl groups. Moreover, BgM is an eumelanin, and its main skeleton has both a benzene ring and an indole, and the branched chain mainly consists of alkanes, alcohols, and fatty acids. BgM was hydrolyzed by H2O2 and four compounds were tentatively analyzed from the UPLC-MS/MS profile. The chemical structure of BgM was characterized as 5,6-dihydroxyindole eumelanin, and the condensed molecular formula is [C28(OR1)4(OR2)3H11O6N4]n.

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We observed homogeneous nucleation process of supercooled liquid Fe by molecular dynamics simulations. Using bond-orientational order parameters together with Voronoi polyhedron method, we characterized local structure, calculated the volume of Voronoi polyhedra of atoms and identified the structure and density fluctuations. We monitored the formation of nucleus and analyzed its inner structure. The birth and growth of the pre-nucleus and nucleus are accompanied with aggregating and disaggregating processes in the time scale of femtosecond. Only the initial solid-like clusters (ISLC), ranging from 1 to 7 atoms, pop up directly from liquid. The relation between the logarithm of number of clusters and the cluster size was found to be linear for ISLCs and was observed to be parabolic for all solid-like clusters (SLC) due to aggregating and disaggregating effects. The nucleus and pre-nuclei mainly consist of body centered cubic (BCC) and hexagonal close packed atoms, while the BCC atoms tend to be located at the surface. Medium-range structure fluctuations induce the birth of ISLCs, benefit the aggregation of embryos and remarkably promote the nucleation. But density fluctuations contribute little to nucleation. The lifetime of most icosahedral-like atoms (ICO) is shorter than 0.7 ps. No obvious relationship was found between structure/density fluctuations and the appearance of ICO atoms.

RT-qPCR remains a vital approach for molecular biology studies aimed at quantifying gene expression in a range of physiological or pathological settings. However, the use of appropriate reference genes is essential to attain meaningful RT-qPCR results. Anastatus japonicus Ashmead (Hymenoptera: Helicopteridae) is an important egg parasitoid wasp and natural enemy of fruit bugs and forest caterpillars. While recent transcriptomic studies have analyzed gene expression profiles in A. japonicus specimens, offering a robust foundation for functional research focused on this parasitoid, no validated A. japonicus reference genes have yet been established, hampering further research efforts. Accordingly, this study sought to address this issue by screening for the most stable internal reference genes in A. japonicus samples to permit reliable RT-qPCR analyses. The utility of eight candidate reference genes (ACTIN, TATA, GAPDH, TUB, RPL13, RPS6, EF1α, RPS3a) was assessed under four different conditions by comparing developmental stages (larvae, pupae, adults), tissues (abdomen, chest, head), sex (male or female adults), or diapause states (diapause induction for 25, 35, 45, or 55 days, or diapause termination). RefFinder was used to calculate gene stability based on the integration of four algorithms (BestKeeper, Normfinder, geNorm, and ΔCt method) to determine the optimal RT-qPCR reference gene. Based on this approach, RPS6 and RPL13 were found to be the most reliable reference genes when assessing different stages of development, while ACTIN and EF1α were optimal when comparing adults of different sexes, RPL13 and EF1α were optimal when analyzing different tissues, and TATA and ACTIN were optimal for different diapause states. These results provide a valuable foundation for future RT-qPCR analyses of A. japonicus gene expression and function under a range of experimental conditions.

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