Circadian patterns and photoperiodic modulation of clock gene expression and neuroendocrine hormone secretion in the marine teleost <i>Larimichthys crocea</i>

The light/dark cycle, known as the photoperiod, plays a crucial role in influencing various physiological activities in fish, such as growth, feeding and reproduction. However, the underlying mechanisms of this influence are not fully understood. This study focuses on exploring the impact of different light regimes (LD: 12 h of light and 12 h of darkness; LL: 24 h of light and 0 h of darkness; DD: 0 h of light and 24 h of darkness) on the expression of clock genes (LcClocka, LcClockb, LcBmal, LcPer1, LcPer2) and the secretion of hormones (melatonin, GnRH, NPY) in the large yellow croaker, Larimichthys crocea. Real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assays were utilized to assess how photoperiod variations affect clock gene expression and hormone secretion. The results indicate that changes in photoperiod can disrupt the rhythmic patterns of clock genes, leading to phase shifts and decreased expression. Particularly under LL conditions, the pineal LcClocka, LcBmal and LcPer1 genes lose their rhythmicity, while LcClockb and LcPer2 genes exhibit phase shifts, highlighting the importance of dark phase entrainment for maintaining rhythmicity. Additionally, altered photoperiod affects the neuroendocrine system of L. crocea. In comparison to the LD condition, LL and DD treatments showed a phase delay of GnRH secretion and an acceleration of NPY synthesis. These findings provide valuable insights into the regulatory patterns of circadian rhythms in fish and may contribute to optimizing the light environment in the L. crocea farming industry.</p

Expression of Tim-3 on TILs in mouse model of transplantable tumor.

<p>6–8-week-old C57BL/6 mice were inoculated with 2×10⁵ B16F0 cells i.d., tumor samples, spleens, and lymph nodes were removed when tumor sizes reached around 15 mm in diameters on day 20. TILs, splenocytes, and lymph node cells were isolated for analysis by flow cytometry. Tim-3 expression on mouse CD4⁺Foxp3⁺ or CD4⁺Foxp3⁻ T cells is shown. Data shown are representative of five independent experiments.</p

Clinical significance of Tim-3⁺ TILs in patients with NSCLC.

<p>Clinical significance of Tim-3⁺ TILs in patients with NSCLC.</p

Three-dimensional (3D) Structural models for B7-H3 proteins were proposed by homology modeling using the crystal structure of known PD-L1.

<p>(A) the model of 2IgB7-H3 (B) the model of 4IgB7-H3.</p

Human 2IgB7-H3 and mouse B7-H3 argument LPS-induced proinflammatory cytokine release.

<p>(A) and (B) is the TNF-α or IL-6 production of human monocytes or mouse monocytes cocultured with various transfectants. (C) Expression of the TNF-α or IL-6 mRNA level in human and mouse monocytes with different transfected cells. β-acin was used as the control. (D) and (E) TNF-α or IL-6 were up-released with the indicated dose of h2IgB7-H3 or mB7-H3 protein.</p

Properties of 4IgB7-H3 in some vertebrates.

<p>The accession number with first letter “E” are Ensembl ID from Ensembl database and GI is from GenBank database.</p

Identification and analysis of B7-H3 isoforms in different species.

<p>(A)phylogenetic analysis of the B7-H3 gene in vertebrates. The tree was constructed from CLUSTAL generated amino acid alignments using the neighbor-joining method. Tree topography was evaluated by bootstrapping 500 times with percentages shown at nodes. The species with duplication event were underlined and the species with only 2IgB7-H3 were labeling with VC after the name. (B) PCR analysis of different RNA samples using B7-H3-specific primer. A product of about 1200 bp corresponds to a 4IgB7-H3 molecule, whereas a 500 bp would represent the 2IgB7-H3 gene. (C) Sequence alignment of deduced translated cow, guinea pig, macaque and dog B7-H3 products. Dark bars or dotted lines above sequence alignment denote exon domains demarcated by genomic sequences.</p

Costimulation of T cell response by h2IgB7-H3 and mB7-H3 while inhibition of T-cell activation by h4IgB7-H3.

<p>(A) Human T cells and mouse T cells were co-cultured with L929 transfectants or CHO transfectants (ratio 10 1) stimulated with plate-bound anti-CD3 mAb and soluble anti-CD28 for 72h. T cell counting was analyzed by cck-8. The data are representative for six independent experiments (B), (C) Culture supernatant was harvested after 72h and subjected to IL-2, IFN-γ measurement.(D) Expression of the IL-2 and IFN-γ mRNA level in human and mouse T cells with different transfected cells. β-acin was used as the control.</p

TIM-3 expression on CD4⁺ TILs and Treg.

<p>TILs were harvested from lung cancer tissue. Cells were then stained for CD4, TIM-3, and FOXP3 (A) or CD4, PD-1, and FOXP3 (B). Lymphocytes were gated for further analysis of CD4 and CD8 T cells. The percentage of each population within CD4⁺ T cell compartment was indicated. Data shown are representative of five independent experiments.</p

TIM-3 is highly expressed on both CD4⁺ and CD8⁺ tumor infiltrating T cells in lung cancer.

<p>Tumor infiltrating lymphocytes (TILs) were harvested from lung cancer tissues, adjacent normal tissues, and peripheral blood monouclear cells (PBMCs) from whole blood of patients. Cells were then stained for CD4, CD8, TIM-3 and PD-1. A. Representative dot-plots show TIM-3 expression on CD4⁺ and CD8⁺ T cells in various tissues from lung cancer patients. Lymphocytes were gated for further analysis of CD4 and CD8 T cells. More than 90% CD4⁺ or CD8⁺ cells were CD3⁺. B. Summarized results of the percentage (%) of TIM-3 expression on CD4⁺ and CD8⁺ T cells from lung cancer patients are shown. Horizontal bars depict the mean percentage of TIM-3 expression on CD4⁺ and CD8⁺ T cells. Error bars: s.e.m. C. Dual expression of TIM-3 and PD-1 on gated CD4⁺ and CD8⁺ T cells is shown. The p-values were calculated using the one-way ANOVA.</p