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Unlike mammals and birds, many fishes have young sex chromosomes, providing excellent models to study sex chromosome differentiation at early stages. Previous studies showed that spotted scat possesses an XX-XY sex determination system. The X has a complete Dmrt3 copy (termed normal) and a truncated copy of Dmrt1 (called Dmrt1b), while the Y has the opposite (normal Dmrt1, which is male-specific, and a truncated Dmrt3 called Dmrt3△-Y). Dmrt1 is the candidate sex determination gene, while the differentiation of other sex-linked genes remains unknown. The spotted scat has proven to be a good model to study the evolution of sex chromosomes in vertebrates. Herein, we sequenced a neighbor gene of this family, Dmrt2, positioned farther from Dmrt1 and closer to Dmrt3 in the spotted scat, and analyzed its sequence variation and expression profiles. The physical locations of the three genes span across an estimated size of >40 kb. The open reading frames of Dmrt2a and its paralog Dmrt2b are 1578 bp and 1311 bp, encoding peptides of 525 and 436 amino acid residues, respectively. Dmrt2a is positioned close to Dmrt3 but farther from Dmrt1 on the same chromosome, while Dmrt2b is not. Sequence analysis revealed several mutations; insertions, and deletions (indels) on Dmrt2a non-coding regions and single-nucleotide polymorphisms (SNPs) on the Dmrt2a transcript. These indels and SNPs are sex-linked and showed high male heterogeneity but do not affect gene translation. The markers designed to span the mutation sites tested on four different populations showed varied concordance with the genetic sexes. Dmrt2a is transcribed solely in the gonads and gills, while Dmrt2b exists in the gonads, hypothalamus, gills, heart, and spleen. The Dmrt2a and Dmrt2b transcripts are profoundly expressed in the male gonads. Analyses of the transcriptome data from five other fish species (Hainan medaka (Oryzias curvinotus), silver sillago (Sillago sihama), Nile tilapia (Oreochromis niloticus), Hong Kong catfish (Clarias fuscus), and spot-fin porcupine fish (Diodon hystrix)) revealed testes-biased expression of Dmrt1 in all, similar to spotted scat. Additionally, the expression of Dmrt2a is higher in the testes than the ovaries in spotted scat and Hainan medaka. The Dmrt2a transcript was not altered in the coding regions as found in Dmrt1 and Dmrt3 in spotted scat. This could be due to the functional importance of Dmrt2a in development. Another possibility is that because Dmrt2a is positioned farther from Dmrt1 and the chromosome is still young, meaning it is only a matter of time before it differentiates. This study undeniably will aid in understanding the functional divergence of the sex-linked genes in fish.

A high-performance liquid chromatography coupled with quadrupole tandem time-of-flight mass (HPLC-QTOF-MS) and ultraviolet spectrometry (HPLC-UV) was established for simultaneous qualitative and quantitative analysis of the major chemical constituents in Caulis Trachelospermi, respectively. The analysis was performed on an Agilent Zorbax Eclipse Plus C18 column (4.6 mm × 150 mm, 5 μm) using a binary gradient system of water and methanol, with ultraviolet absorption at 230 nm. Based on high-resolution ESI-MS/MS fragmentation behaviors of the reference standards, the characteristic cleavage patterns of lignano-9, 9'-lactones and lignano-8'-hydroxy-9, 9'-lactones were obtained. The results demonstrated that the characteristic fragmentation patterns are valuable for identifying and differentiating lignano-9,9'-lactones and lignano-8'-hydroxy-9,9'-lactones. As such, a total of 25 compounds in Caulis Trachelospermi were unambiguously or tentatively identified via comparisons with reference standards or literature. In addition, 14 dibenzylbutyrolatone lignans were simultaneously quantified in Caulis Trachelospermi by HPLC-UV method. The method is suitable for the qualitative and quantitative analyses of dibenzylbutyrolatone lignans in Caulis Trachelospermi.

Initial investigation for new active herbal extract with inhibiting activity on JAK/STAT signaling pathway revealed that the extract of Caulis Trachelospermi, which was separated by 80% alcohol extraction and subsequent HP-20 macroporous resin column chromatography, was founded to strongly inhibit IFN-γ-induced STAT1-responsive luciferase activity (IFN-γ/STAT1) with IC50 value of 2.43 μg/mL as well as inhibiting IL-6-induced STAT3-responsive luciferase activity (IL-6/STAT3) with IC50 value of 1.38 μg/mL. Subsequent study on its active components led to the isolation and identification of two new dibenzylbutyrolactone lignans named 4-demethyltraxillaside (1) and nortrachelogenin 4-O-β-d-glucopyranoside (2), together with six known compounds. The lignan compounds 1–4 together with other lignan compounds isolated in previous study were tested the activities on IFN-γ/STAT1 and IL-6/STAT3 pathways. The following result showed that the main components trachelogenin and arctigenin had corresponding activities on IFN-γ/STAT1 pathway with IC50 values of 3.14 μM and 9.46 μM as well as trachelogenin, arctigenin and matairesinol strongly inhibiting IL-6/STAT3 pathway with IC50 values of 3.63 μM, 6.47 μM and 2.92 μM, respectively.