Investigation into the association of FNDC1 and ADAMTS12 gene expression with plumage coloration in Muscovy ducks.

To elucidate the molecular genetic mechanisms underpinning feather color in Muscovy ducks. A cohort of 100 Muscovy ducks was meticulously selected for this research. Follicular tissues from ducks exhibiting black and white plumage served as the experimental samples. From these tissues, RNA and proteins were extracted for further analysis. The RNA underwent reverse transcription polymerase chain reaction amplification, followed by validation through western blot assays. The data revealed a significant upregulation in the expression of FN domain-containing protein 1 (FNDC1) and ADAMTS12 genes in Muscovy ducks with white plumage traits as opposed to those with black plumage traits. Specifically, individuals with pure white plumage demonstrated a markedly elevated expression of the FNDC1 gene in comparison to their pure black counterparts. Conversely, expression levels of the ADAMTS12 gene were found to be reduced in ducks with pure black plumage relative to those with pure white plumage. Notably, the expression patterns of FNDC1 and ADAMTS12 genes exhibited inconsistencies between mRNA and protein levels. This study offers significant insights into the molecular genetic mechanisms underlying feather color variation in Muscovy ducks. FNDC1 and ADAMTS12 could be considered potential targets for genetic manipulation or selective breeding strategies aimed at achieving specific feather color phenotypes in Muscovy ducks.

Exploration of the genetic influence of MYOT and MB genes on the plumage coloration of Muscovy ducks.

Plumage color, a pivotal attribute delineating diverse Muscovy duck strains, assumes considerable significance within the field of Muscovy duck breeding research. This study extends the existing research by delving into the hereditary aspects of genes associated with plumage coloration in Muscovy ducks. The principal objective is to discern marker genes conducive to targeted breeding strategies based on plumage color, thereby furnishing indispensable technical foundations for the development of novel Muscovy duck varieties. Our investigation focused on scrutinizing the impact of MYOT and MB genes on the genetic expression of plumage color at both the RNA and protein levels in Muscovy ducks. The results elucidate that black Muscovy ducks manifest markedly elevated mRNA and protein expression levels of MYOT and MB genes in comparison to their white counterparts, indicating that both genes may play a constructive regulatory role in the context of plumage coloration in Muscovy ducks. The outcomes of this study delineate a discernible correlation between MYOT and MB genes and the plumage coloration in Muscovy ducks. Employing gene expression analysis, we successfully identified candidate genes that may be intricately linked to the determination of plumage color in these ducks.

Multiple stimulus-response berberine plus baicalin micelles with particle size-charge-release triple variable properties for breast cancer therapy.

OBJECTIVE: The aim was to develop a nanoscale drug delivery system with enzyme responsive and acid sensitive particle size and intelligent degradation aiming to research the inhibitory effect on breast cancer. SIGNIFICANCE: The delivery system addressed the problems of tissue targeting, cellular internalization, and slow drug release at the target site, which could improve the efficiency of drug delivery and provide a feasible therapeutic approach for breast cancer. METHODS: The acid sensitive functional material DSPE-PEG2000-dyn-PEG-R9 was synthesized by Michael addition reaction. Then, the berberine plus baicalin intelligent micelles were prepared by thin-film hydration. Subsequently, we characterized the physical and chemical properties of berberine plus baicalin intelligent micelles, evaluated its anti-tumor effects in vivo and in vitro. RESULTS: The target molecule was successfully synthesized, and the intelligent micelles showed excellent chemical and physical properties, delayed drug release and high encapsulation efficiency. In vitro and in vivo experiments also confirmed that the intelligent micelles could effectively target tumor sites, penetrate tumor tissues, enrich in tumor cells, inhibit tumor cell proliferation, inhibit tumor cell invasion and migration, and induce tumor cell apoptosis. CONCLUSION: Berberine plus baicalin intelligent micelles have excellent anti-tumor effects and no toxicity to normal tissues, which provides a new potential drug delivery strategy for the treatment of breast cancer.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing.

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P<0.05), and RSV did not significantly increase the sperm motility (0.44 vs. 0.40, P>0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨm. RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 µmol/L vs. 0.25 µmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.

The CD8α gene in duck (Anatidae): cloning, characterization, and expression during viral infection.

Cluster of differentiation 8 alpha (CD8α) is critical for cell-mediated immune defense and T-cell development. Although CD8α sequences have been reported for several species, very little is known about CD8α in ducks. To elucidate the mechanisms involved in the innate and adaptive immune responses of ducks, we cloned CD8α coding sequences from domestic, Muscovy, Mallard, and Spotbill ducks using reverse transcription polymerase chain reaction (RT-PCR). Each sequence consisted of 714 nucleotides and encoded a signal peptide, an IgV-like domain, a stalk region, a transmembrane region, and a cytoplasmic tail. We identified 58 nucleotide differences and 37 amino acid differences among the four types of duck; of these, 53 nucleotide and 33 amino acid differences were between Muscovy ducks and the other duck species. The CD8α cDNA sequence from domestic duck consisted of a 61-nucleotide 5' untranslated region (UTR), a 714-nucleotide open reading frame, and an 849-nucleotide 3' UTR. Multiple sequence alignments showed that the amino acid sequence of CD8α is conserved in vertebrates. RT-PCR revealed that expression of CD8α mRNA of domestic ducks was highest in the thymus and very low in the kidney, cerebrum, cerebellum, and muscle. Immunohistochemical analyses detected CD8α on the splenic corpuscle and periarterial lymphatic sheath of the spleen. CD8α mRNA in domestic ducklings was initially up-regulated, and then down-regulated, in the thymus, spleen, and liver after treatment with duck hepatitis virus type I (DHV-1) or the immunostimulant polyriboinosinic polyribocytidylic acid (poly I:C).

Molecular cloning and expression analysis of ferritin, heavy polypeptide 1 gene from duck (Anas platyrhynchos).

H-ferritin is a core subunit of the iron storage protein ferritin, and is related to the pathogenesis of malignant diseases. A differential expressed sequence tag of the ferritin, heavy polypeptide 1 gene (FTH1) was obtained from our previously constructed suppression subtractive cDNA library from 3-day-old ducklings challenged with duck hepatitis virus type I (DHV-1). The expression and function of FTH1 in immune defense against infection remains largely unknown in ducks. In this study, the full-length duFTH1 cDNA was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. It consisted of 153 basepairs (bp) 5'untranslated region (UTR), 183 bp 3'UTR, and 546 bp open reading frame that encodes a single protein of 181 amino acid residues. duFTH1 shares high similarity with FTH1 genes from other vertebrates. The amino acid sequence possesses the conserved domain of typical ferritin H subunits, including seven metal ligands in the ferroxidase center, one iron binding region signature, and a potential bio-mineralization residue (Thy(29)). Moreover, in agreement with a previously reported ferritin H subunit, we identified an iron response element in the 5'UTR. RT-PCR analyses revealed duFTH1 mRNA is widely expressed in various tissues. Real-time quantitative polymerase chain reaction analyses suggested that duFTH1 mRNA is significantly up-regulated in the liver after DHV-1 injection or polyriboinosinic polyribocytidylic acid (polyI:C) treatment, reaching a peak 4 h post-infection, and dropping progressively and returning to normal after 24 h. Our findings suggest that duFTH1 functions as an iron chelating protein subunit in duck and contributes to the innate immune responses against viral infections.

DNA methylation and regulation of the CD8A after duck hepatitis virus type 1 infection.

BACKGROUND: Cluster of differentiation 8 (CD8) is expressed in cytotoxic T cells, where it functions as a co-receptor for the T-cell receptor by binding to major histocompatibility complex class I (MHCI) proteins, which present peptides on the cell surface. CD8A is critical for cell-mediated immune defense and T-cell development. CD8A transcription is controlled by several cis-acting elements and trans-acting elements and is also regulated by DNA methylation. However, the epigenetic regulation of CD8A in the duck and its relationship with virus infection are still unclear. RESULTS: We investigated the epigenetic transcriptional regulatory mechanisms, such as DNA methylation, for the expression of the CD8A and further evaluated the contribution of such epigenetic regulatory mechanisms to DHV-I infection in the duck. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed the highest level of CD8A expression to be in the thymus, followed by the lungs, spleen, and liver, and the levels of CD8A expression were very low in the kidney, cerebrum, cerebellum, and muscle in the duck. RT-qPCR also demonstrated that the CD8A mRNA was down-regulated significantly in morbid ducklings treated with DHV-1 and up-regulated significantly in non-morbid ducklings in all the tissues tested. In addition, hypermethylation of CD8A was detected in the morbid ducklings, whereas relatively low methylation of CD8A was evident in the non-morbid ducklings. The CD8A mRNA level was negatively associated with the CpG methylation level of CD8A and global methylation status. CONCLUSIONS: We concluded that the mRNA level of the CD8A was negatively associated with the CpG methylation level of CD8A and global methylation status in the duck, suggesting that the hypermethylation of CD8A may be associated with DHV-1 infection. The first two CpG sites of the CD8A promoter region could be considered as epigenetic biomarkers for resistance breeding against duckling hepatitis disease in the duck.

Identification and expression analysis of the leukocyte cell-derived chemotaxin-2 (LECT2) gene in duck (Anas platyrhynchos).

Leukocyte cell-derived chemotaxin 2 (LECT2), first identified as a chemotactic factor, is involved in the regulation of liver regeneration, carcinogenesis, and natural killer T-cell homeostasis in mammals. The function of LECT2 in the duck remains unclear, however. A suppression subtractive cDNA library was constructed from the livers of 3-day-old ducklings treated with duck hepatitis virus type I (DHV-1). A total of 66 expressed sequence tags (ESTs) were identified in the libraries. Among the novel gene fragments identified was the LECT2 gene. Full-length duck LECT2 (duLECT2) complementary DNA (cDNA) was obtained using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). The cDNA consisted of a 50 nucleotide 5' untranslated region (UTR), an 84 nucleotide 3' UTR, and a 1020 nucleotide open reading frame encoding a single protein of 339 amino acids. In agreement with a previously reported LECT2 sequence, the predicted amino acid sequence contains characteristic phosphorylation and N-glycosylation sites. DuLECT2 is highly similar to LECT2 genes from other vertebrates. Phylogenetic analysis demonstrated that the LECT2 gene has been highly conserved throughout vertebrate evolution. RT-PCR analyses revealed that duLECT2 mRNA is widely expressed in healthy tissues. They also showed that duLECT2 mRNA is significantly up-regulated in the liver and spleen following injection with DHV-1 or polyriboinosinic polyribocytidylic acid (poly I:C), peaking 4 or 12h post-challenge in the liver and spleen, respectively, and afterwards gradually returning to normal. Our findings suggest that duLECT2 contributes to the innate immune response against viral infections.

[Identification and genetic analysis of SNPs in duck adiponectin gene].

Single nucleotide polymorphisms (SNPs) within the duck adiponectin gene were detected by single strand conformation polymorphism (SSCP) using 5 pairs of primers to amplify an area spanning the open reading frame. Eight duck breeds, including Kunshan Sheldrake, Cherry Valley Meat duck, Gaoyou duck, Shanma duck, Jinding duck, Longbai duck, Jingjiang Sheldrake and White feather Muscovy duck, were used. Seven nucleotide variations were found, of which G430A, A457G, and T523C resulted in amino acid changes of A144T, I153V, and Y175H, respectively. The remaining 4 SNPs were C507T, T540C, C576T and C597T. Eight genotypes (AA, AB, AC, BB, BC, CC, DD, and DE) were detected in the 8 breeds. Chi(2) analysis showed that the distribution of the eight genotypes was very different among the different breeds (P < 0.01). Ex-cept for the Jingding duck, all breeds were in accordance with the Hardy-Weinberg equilibrium. Genetic analysis indicated that homozygosity was highest in the Jinding duck, lowest in the Gaoyou duck and similar in other breeds. Polymorphism information content (PIC) was low in the Jinding, high in the Gaoyou and intermediate in other breeds. These results showed that the adiponectin gene had a high level of polymorphism in different duck breeds, and could be used as a candidate gene to analyze the correlation between its polymorphism and fat traits in duck.