Screening of Reference Genes for Quantitative Real-Time PCR Analysis in Tissues and during Testis Development, and Application to Analyze the Expression of kifc1 in Hemibarbus labeo (Teleostei, Cypriniformes, Cyprinidae).

The selection of proper reference genes is vital for ensuring precise quantitative real-time PCR (qPCR) assays. This study evaluates the stability of the expression of nine candidate reference genes in different tissues and during testicular development in H. labeo. The results show that eef1a is recommended as a reference gene for qPCR analysis in tissues and during testicular development. Furthermore, we evaluated the optimal number of reference genes needed when calculating gene expression levels using the geomean method, revealing that two reference genes are sufficient. Specifically, eef1a and rps27 are recommended for analysis of gene expression in tissues, whereas eef1a and actb are advised for evaluating gene expression during testicular development. In addition, we examined the expression pattern of kifc1, a kinesin involved in the reshaping of spermatids. We detected peak expression levels of kifc1 in testes, with its expression initially increasing before decreasing throughout testicular development. The highest expression of kifc1 was observed in stage IV testes, the active period of spermiogenesis, suggesting a possible role for kifc1 in the regulation of the reshaping of spermatids and hence testicular development. This study represents the first investigation of reference genes for H. labeo, providing a foundation for studying gene expression patterns and investigating gene expression regulation during testicular development.

Histological and Transcriptomic Insights into the Ovary Development of Hemibarbus labeo Injected with Spawn-Inducing Hormones.

Naturally, the ovaries of many farmed fish can only develop to stage IV (mainly including stage IV oocytes, known as full-grown postvitellogenic oocytes). Therefore, spawn-inducing hormone injections are used to promote ovary development and oocyte maturation, facilitating reproduction in the aquaculture industry. The study of spawn-inducing hormones and their underlying neuroendocrine mechanisms has been a recent focus in fish reproductive biology. However, the intra-ovarian regulatory mechanisms of ovary development and oocyte maturation after hormone injection require further investigation. In this study, we explored the histological and transcriptomic map of the ovary of Hemibarbus labeo after hormone injection to reveal changes in the ovary. The gonad index significantly increased after hormone injection for 5.5 h, after which no significant change was observed. Histological analysis showed that the nuclei had moved to one side of the oocytes at 5.5 h after hormone injection. Moreover, the volume of the oocytes increased and their yolk membranes thickened. Oocytes then underwent their first meiotic division at 5.5-11 h after hormone injection. Subsequently, the follicular membrane was ruptured, and ovulation was completed at 11-16.5 h after hormone injection. In addition, we identified 3189 differentially expressed genes (DEGs) on comparing the transcriptomes at different time points after hormone injection. These DEGs were significantly enriched in the GO terms of nervous system process, molecular transducer activity, and extracellular region, and the KEGG pathways of TNF signaling and cytokine-cytokine receptor interaction; these may play important roles in ovary development and oocyte maturation. Within these pathways, genes such as apoe, creb3, jun, junb, il11, and il8 may play important roles in steroid hormone synthesis and ovulation. Conclusively, our results show detailed sequential dynamics of oocyte development and provide new insights into the intra-ovarian regulatory mechanisms of ovarian development and oocyte maturation in H. labeo. These findings may be important for research on improving egg quality and reproduction in aquaculture.

Functional insights of digestion, absorption, and immunity in different segments of the intestine in Hemibarbus labeo from transcriptomic analysis.

The intestine is an important organ for food digestion and absorption and body immunity in fish. In this study, we investigated the abundance of transcripts from different segments of the intestinal tract using transcriptome sequencing technology in Hemibarbus labeo, to provide functional insights into digestion, absorption, and immunity in the anterior intestine (AI), middle intestine (MI), and posterior intestine (PI). We found 5646 differentially expressed genes (DEGs), which were significantly enriched to GO terms of carbohydrate metabolic process, transmembrane transport, iron ion binding, lipid metabolic process, and KEGG pathway of fat digestion and absorption, mineral absorption, protein digestion and absorption, vitamin digestion and absorption, indicating that the digestion and absorption function of food is different in AI, MI, and PI. In practice, most genes, enriched in the KEGG pathway for digestion and absorption of nutrients, are upregulated in AI and MI, indicating stronger roles for food digestion and absorption in these segments. Furthermore, we found that genes involved in the KEGG pathway of lysosome and endocytosis pathway are upregulated in PI, suggesting stronger antigen-presenting capabilities in PI. However, some cytokine receptor genes, including ccr4, cxcr2, tnfrsf9, il6r, csf3r, and cxcr4, are highly expressed in AI, reflecting the regional immune specialization in different segments. This study provides functional insights into digestion, absorption, and immunity in different segments of the intestine and supports the regional functional specialization within different segments of the intestine in H. labeo.

Psychometric Evaluation of the Chinese Version of Mild Cognitive Impairment Questionnaire among Older Adults with Mild Cognitive Impairment.

BACKGROUND: There is a lack of instruments for measuring quality of life (QOL) in Chinese patients with mild cognitive impairment (MCI). This study aimed to translate the Mild Cognitive Questionnaire (MCQ) into the Chinese language and to evaluate the reliability and construct validity of the MCQ-Chinese among older adults with MCI. METHODS: Linguistic translation and validation of the questionnaire were conducted according to the MCQ developer and Oxford University Innovation guidelines. After a pilot test, the final version of the MCQ-Chinese was applied to a convenience sample of older adults with MCI (n = 186). Cronbach's alpha and confirmatory factor analyses were used to assess the reliability and construct validity of the MCQ-Chinese. In addition, non-parametric analysis was used to assess convergent and discriminant validity. RESULTS: The total scale and all the factors had good internal consistency, with Cronbach's alpha values ranging from 0.90 to 0.92. Confirmatory factor analysis indicated satisfactory goodness of fit for the 2-factor MCQ. The MCQ-Chinese had a good convergent validity, and the discriminant validity was confirmed with a significant difference in MCQ scores in different health conditions. CONCLUSIONS: MCQ-Chinese is a reliable tool for assessing QOL among Chinese older adults with MCI.

Host defense peptide LEAP-2 contributes to monocyte/macrophage polarization in barbel steed (Hemibarbus labeo).

The liver-expressed antimicrobial peptide 2 (LEAP-2) plays a vital role in host immunity against pathogenic organisms. In the present study, cDNA of the LEAP-2 gene was cloned and sequenced from the barbel steed (Hemibarbus labeo). The predicted amino acid sequence of the barbel steed LEAP-2 comprises a signal peptide and a prodomain, which is followed by the mature peptide. Sequence analysis revealed that barbel steed LEAP-2 belongs to the fish LEAP-2A cluster and that it is closely related to zebrafish LEAP-2A. We found that barbel steed LEAP-2 transcripts were expressed in a wide range of tissues, with the highest mRNA levels detected in the liver. In response to lipopolysaccharide (LPS) treatment, LEAP-2 was significantly upregulated in the liver, head kidney, spleen, gill, and mid intestine. A chemically synthesized LEAP-2 mature peptide exhibited selective antimicrobial activity against several bacteria in vitro. Moreover, LEAP-2, alone or in combination with LPS or phorbol 12-myristate 13-acetate, strongly induced a pro-inflammatory reaction in barbel steed monocytes/macrophages (MO/MФ), involving the induction of iNOS activity, respiratory burst, and the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-1ß. Collectively, the results of this study indicate the importance of fish LEAP-2 in the M1-type polarization of MO/MΦ.

Molecular characterization of a hepcidin homologue in starry flounder (Platichthys stellatus) and its synergistic interaction with antibiotics.

Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in host immunity against pathogenic organisms. Most fish hepcidins exert bactericidal activities against a wide range of pathogens. In this study, we identified a cDNA sequence encoding a hepcidin homologue (PsHepcidin) in the starry flounder Platichthys stellatus. The predicted amino acid sequence of PsHepcidin comprises a signal peptide and a prodomain, which are followed by the mature peptide. Sequence analysis revealed that PsHepcidin belongs to the fish HAMP2 cluster and that it is closely related to mudskipper hepcidin-2. Expression of PsHepcidin mRNA was detected in all examined immune-related tissues, with the highest transcript levels being found in the liver. In response to lipopolysaccharide treatment, PsHepcidin was significantly up-regulated in the liver, kidney, and spleen in a time-dependent manner. Chemically synthesized mature peptides of PsHepcidin were found to exhibit broad antimicrobial activity in vitro. We also investigated the combined effect of PsHepcidin and conventional antibiotics and found that these combinations showed synergistic effects against most of the examined bacterial strains. Collectively, the results of this study indicate that PsHepcidin exhibits potent antibacterial activity both independently and when used in combination with conventional antibiotics.