Application effectiveness of "Youxin-1" in genetic diversity and structure analysis of local chickens.

China's local chicken breeds are rich in resources, and have formed different germplasm characteristics in the process of long-term selection and evolution. Scientific assessment of population genetic diversity and identification of inter-breed genetic structure are of great value to the protection and innovative utilization of local chicken breed resource. In order to evaluate the application effectiveness of 23K SNP chip "Youxin-1" in the analysis of genetic diversity and genetic structure of local chickens, we used RADseq to identify genomic genetic variation of 21 local chicken breeds and developed 23K chip "Youxin-1". The genetic statistics of each variety were calculated based on two sets of SNP data, and correlation, fitting and phylogenetic analysis were carried out to evaluate the application effectiveness of the chip. The results showed that the observed heterozygosity (Ho), polymorphism information content (PIC), inbred coefficient (FROH) and genetic differentiation coefficient (Fst) calculated based on the two SNP data sets were basically consistent in the 21 local chicken breeds. The genetic diversity of Langya chicken (LA), Piao chicken (PJ) and Wenchang chicken (WC) was relatively rich. The genetic diversity of Bian chickens (BJ), Langshan chickens (LS), Gushi chickens (GS), Dongxiang blue-eggshell chickens (DX) and Beijing fatty chickens (BY) was relatively poor, and the correlation coefficients of Ho, PIC, FROH and average Fst in the two groups were 0.794, 0.901, 0.926 and 0.984, respectively, all reaching extremely significant levels (P<0.01) with a high degree of fit (P<0.001) and R2 were 0.644, 0.827, 0.916 and 0.927. For the two sets of SNP data, the evolutionary tree constructed by neighbor-joining (NJ) method and maximum likelihood (ML) method was reasonable, and the 21 local chicken breeds were generally divided into six categories, which was consistent with the formation history and geographical distribution of the varieties. The 23K chip also realized reasonable clustering of the five new varieties without individual deviation. There are some differences in the estimation of genetic statistics using SNP with different densities, and data standardization is needed. 23K chip has good efficacy in the analysis of genetic diversity and structure of local chickens.

[Construction of three HLA-A*0201-peptide tetramers and their preliminary application in detection of vitiligo-specific cytotoxic T lymphocytes].

OBJECTIVE: To construct vitiligo-specific HLA-A*0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxic T lymphocytes (CTL). METHODS: Proteins HLA-A0201*-BSP and ß2M were obtained by effective prokaryotic expression. The purified proteins were refolded with vitiligo antigen peptides MelanA 26-35, gp100 209-217, and tyrosinase 1-9, respectively to form HLA-A*0201-peptide complex. The complex was biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with phycoerythrin (PE)-streptavidin at a ratio of 4∶1 and identified by Dot-blot assay. The capacity of tetramer to detect vitiligo-specific CTL was analyzed by flow cytometry. RESULTS: The biotinylation of vitiligo-specific HLA-A*0201-peptide tetramers were successfully performed by Dot-blot. Flow cytometry analysis indicated that the tetramer effectively bound to specific CTL from peripheral blood of patients with vitiligo. CONCLUSION: Three kinds of biotinylated vitiligo-specific HLA-A*0201-peptide tetramers have been constructed successfully. The tetramer can detect antigen specific CTL from patients with vitiligo.

Metagenomic Sequencing Revealed Differences in the Composition of Cecal Microbes in Different Breeds of Chickens.

Intestinal microorganisms are closely related to the health, development, and production performance of animals and can also provide basic information for us to fully understand and explore the germplasm characteristics and traits of local chicken breeds. In this experiment, we used five local Chinese chicken breeds as research materials; metagenomic sequencing technology was used to explore the differences in the composition of intestinal microorganisms in different breeds, and it was found that each local chicken breed had unique unigenes, of which Silkies had the most, with a total of 12,948. GO and KEGG analyses found that the biological processes most enriched by differential functional genes include genetic coding, macromolecular transport, protein synthesis, and molecular functions such as glycoprotein binding, protein hydrolysis, etc. Each breed is enriched with specific pathways, such as Anyi tile-like gray chickens, which are enriched with pathways related to disease resistance, while Gamecocks' enrichment is related to amino acid metabolism. Random Forest and LEfSe analyses revealed specific species of intestinal microorganisms in the cecum of different breeds, such as Exiguobacterium, which is associated with melanin deposition in Silkies. Therefore, we infer that gut microorganisms are closely related to the formation of chicken breed characteristics, and the results of this experiment can provide a theoretical basis for the discovery of high-quality traits and the improvement of feeding conditions in the future.

Independent and combined effects of antibiotic stress and EM microbial agent on the nitrogen and humus transformation and bacterial community successions during the chicken manure composting.

This study aimed to investigate the independent and combined effects of antibiotic and EM microbial agent on the nitrogen and humus (HS) transformations as well as the bacterial community successions during the chicken manure and rice husk composting. EM microbial agent accelerated the oxytetracycline (OTC) degradation, but slowed down the norfloxacin (NOR) degradation. OTC inhibited the TN retention and promoted the HS accumulation, both NOR and EM microbial agent inhibited the TN retention and HS accumulation, while EM microbial agent showed an antagonistic effect on TN immobilization with antibiotics and reduced the impacts of antibiotics on HS. Obvious bacterial community successions occurred. Firmicutes were related to HS transformation, while Firmicutes, Actinobacteriota and Proteobacteria were associated with nitrogen conversion. NOR promoted the transformations of NH4+-N to NO3--N and FA to HA. The findings provided theoretical data for the recycle of antibiotic-contaminated manure and the efficient production of high-quality compost.

Expression and significance of mammalian target of rapamycin in cutaneous squamous cell carcinoma and precancerous lesions.

The aim of this study is to explore the role of mammalian target of rapamycin (mTOR) in cutaneous squamous cell carcinoma (CSCC), Bowen's disease (BD), and actinic keratosis (AK) with squamous cell differentiation abnormality and its relationship with the degree of tumor proliferation. Thirty cases of clinical paraffin specimens of CSCC, BD, and AK were each collected from Jinhua Fifth Hospital, while 30 cases of normal skin specimens surgically resected in Department of Plastic Surgery were selected as controls. The expressions of mTOR and Ki-67 in tissues were detected by immunohistochemical staining. The positive expression rate of mTOR in the CSCC group was higher than those in the BD group and AK group (P < 0.05), while it was higher in the BD group and AK group than in the normal skin group (P < 0.05). The CSCC group had a higher positive expression rate of Ki-67 than the AK group (P < 0.01). The results of logistic regression analysis showed that the pathogenic site [odds ratio (OR) = 1.189, 95% confidence interval (95%CI): 1.028-1.381, P = 0.021], course of disease (OR = 2.059, 95%CI: 1.036-4.087, P = 0.043), and differentiation degree (OR = 1.325, 95%CI: 1.169-1.512, P = 0.001) were independent factors for the positive expression of mTOR. OR>1, indicating that the factor is a risk factor. The expression levels of mTOR in CSCC, BD, and AK were positively correlated with the expression level of Ki-67 (r = 0.827, P < 0.01, r = 0.608, P < 0.01, r = 0.368, P = 0.045). These results suggest that mTOR may be involved in the pathogenesis of CSCC, and related to the proliferation degree of CSCC, as an index reflecting the proliferation status of CSCC.

Online preconcentration in capillary electrophoresis with contactless conductivity detection for sensitive determination of sorbic and benzoic acids in soy sauce.

A sensitive method of online preconcentration followed by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4)D) is evaluated as a novel approach for the determination of benzoic acid and sorbic acid in soy sauce. The online preconcentration technique, namely field-enhanced sample injection, coupled with CE-C(4)D were successfully developed and optimized. In order to reduce the complex matrix interference resulting from the constituents of soy sauce, a suitable sample clean-up procedure was also investigated for real sample pretreatment. Under optimized conditions, sorbic acid and benzoic acid were well separated within 10 min, and the detection limits were 0.05 µM (5.6 µg L(-1)) and 0.08 µM (9.8 µg L(-1)), respectively. The accuracy was tested by spiking 10.0 mg L(-1) and 100.0 mg L(-1) of standards in the soy sauce samples, and the recoveries were 95-99%, respectively. Results of this study show a great potential for the proposed method as a tool for the fast screening of benzoic acid and sorbic acid in a complex matrix.