Integrated analysis of the DNA methylome and RNA transcriptome during the development of skeletal muscle in Duroc pigs.

BACKGROUND: Skeletal muscle development plays a crucial role in yield and quality of pork; however, this process is influenced by various factors. In this study, we employed whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing to comprehensively investigate the longissimus dorsi muscle (LDM), aiming to identify key genes that impact the growth and development of Duroc pigs with different average daily gains (ADGs). RESULTS: Eight pigs were selected and divided into two groups based on ADGs: H (774.89 g) group and L (658.77 g) group. Each pair of the H and L groups were half-siblings. The results of methylation sequencing revealed 2631 differentially methylated genes (DMGs) involved in metabolic processes, signalling, insulin secretion, and other biological activities. Furthermore, a joint analysis was conducted on these DMGs and the differentially expressed genes (DEGs) obtained from transcriptome sequencing of the same individual. This analysis identified 316 differentially methylated and differentially expressed genes (DMEGs), including 18 DMEGs in promoter regions and 294 DMEGs in gene body regions. Finally, LPAR1 and MEF2C were selected as candidate genes associated with muscle development. Bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qRT-PCR) revealed that the promoter region of LPAR1 exhibited significantly lower methylation levels (P < 0.05) and greater expression levels (P < 0.05) in the H group than in the L group. Additionally, hypermethylation was observed in the gene body region of MEF2C, as was a low expression level, in the H group (P < 0.05). CONCLUSIONS: These results suggest that the differences in the ADGs of Duroc pigs fed the same diet may be influenced by the methylation levels and expression levels of genes related to skeletal muscle development.

Overexpression of CuZn superoxide dismutase improves high-density lipoprotein function in swine.

Cardiovascular disease (CVD) has been the leading cause of death worldwide. As a chronic inflammatory disease, atherosclerosis (AS) acts as the initiating factor for CVD and reactive oxygen species (ROS) play a vital role in its development. Superoxide dismutases (SOD) can alleviate the detrimental effects of ROS and serve as the first line of defense through detoxifying the products derived from oxidative stress in vivo. Considering the potential preventive effects of high-density lipoprotein (HDL) on AS and the close relationship between CuZn superoxide dismutase (CuZnSOD) and HDL, the present work investigated whether CuZnSOD overexpression in swine could improve the function of HDL. Seven CuZnSOD transgenic swine, constructed by sperm and magnetic nanoparticles, demonstrated overexpressed CuZnSOD in the liver (P < 0.01) but comparable level to control in plasma (P > 0.05). CuZnSOD overexpression significantly down-regulated the levels of triglyceride (TG), apolipoprotein A-I (apoA-I) (P < 0.05), and high-density lipoprotein cholesterol (HDL-C) (P < 0.01) in plasma. In the presence of CuZnSOD overexpression, HDL3 significantly inhibited levels of IL-6 and TNF-α induced by oxidized low-density lipoprotein (oxLDL) (P < 0.05), indicating enhanced anti-inflammatory activity of HDL. At the same time, HDL-mediated cholesterol efflux did not decrease (P > 0.05). CuZnSOD overexpression improves the anti-inflammatory function of HDL despite decreased levels of HDL-C. In Conclusion, CuZnSOD overexpression improves HDL function in swine.

Full-Length Transcriptome Analysis of Skeletal Muscle of Jiangquan Black Pig at Different Developmental Stages.

Skeletal muscle grows in response to a combination of genetic and environmental factors, and its growth and development influence the quality of pork. Elucidating the molecular mechanisms regulating the growth and development of skeletal muscle is of great significance to both animal husbandry and farm management. The Jiangquan black pig is an excellent pig breed based on the original Yimeng black pig, importing the genes of the Duroc pig for meat traits, and cultivated through years of scientific selection and breeding. In this study, full-length transcriptome sequencing was performed on three growth stages of Jiangquan black pigs, aiming to study the developmental changes in Jiangquan black pigs at different developmental stages at the molecular level and to screen the key genes affecting the growth of skeletal muscle in Jiangquan black pigs. We performed an enrichment analysis of genes showing differential expression and constructed a protein-protein interaction network with the aim of identifying core genes involved in the development of Jiangquan black pigs. Notably, genes such as TNNI2, TMOD4, PLDIM3, MYOZ1, and MYH1 may be potential regulators of muscle development in Jiangquan black pigs. Our results contribute to the understanding of the molecular mechanisms of skeletal muscle development in this pig breed, which will facilitate molecular breeding efforts and the development of pig breeds to meet the needs of the livestock industry.

Innate immune responses to duck Tembusu virus infection.

The disease caused by duck Tembusu virus (DTMUV) is characterized by severe egg-drop in laying ducks. Currently, the disease has spread to most duck-raising areas in China, leading to great economic losses in the duck industry. In the recent years, DTMUV has raised some concerns, because of its expanding host range and increasing pathogenicity, as well as the potential threat to public health. Innate immunity is crucial for defending against invading pathogens in the early stages of infection. Recently, studies on the interaction between DTMUV and host innate immune response have made great progress. In the review, we provide an overview of DTMUV and summarize current advances in our understanding of the interaction between DTMUV and innate immunity, including the host innate immune responses to DTMUV infection through pattern recognition receptors (PRRs), signaling transducer molecules, interferon-stimulated genes (ISGs), and the immune evasion strategies employed by DTMUV. The aim of the review is to gain an in-depth understanding of DTMUV pathogenesis to facilitate future studies.

Expression of lipid metabolism genes provides new insights into intramuscular fat deposition in Laiwu pigs.

OBJECTIVE: The objective of this study was to measure the special expression pattern of lipid metabolism genes and investigate the molecular mechanisms underlying intramuscular fat (IMF) deposition in Longissimus dorsi muscle of Laiwu pigs. METHODS: Thirty-six pigs (Laiwu n = 18; Duroc×Landrace×Yorkshire n = 18) were used for the measurement of the backfat thickness, marbling score, IMF content, and expression of lipid metabolism genes. RESULTS: Significant correlations were found between IMF content and the mRNA expression of lipid metabolism genes. Of the 14 fat deposition genes measured, fatty acid synthase (FASN) showed the strongest correlation (r = 0.75, p = 0.001) with IMF content, and of the 6 fat removal genes, carnitine palmitoyl transferase 1B (CPT1B) exhibited the greatest negative correlation (r = -0.66, p = 0.003) with IMF content in Laiwu pig. Multiple regression analysis showed that CPT1B, FASN, solute carrier family 27 member 1 (SLC27A1), and fatty acid binding protein 3 (FABP3) contributed 38% of the prediction value for IMF content in Laiwu pigs. Of these four variables, CPT1B had the greatest contribution to IMF content (14%) followed by FASN (11%), SLC27A1 (9%), and FABP3 (4%). CONCLUSION: Our results indicate that the combined effects of an upregulation in fat deposition genes and downregulation in fat removal genes promotes IMF deposition in Laiwu pigs.

Overexpression of PID1 reduces high density lipoprotein level and functionality in swine.

Phosphotyrosine interaction domain 1 (PID1), a protein with a phosphotyrosine-binding (PTB) domain, interacts with the lipoprotein receptor-related protein 1 (LRP1) to reduce the insulin sensitivity of adipocyte. Considering the role of LRP1 in lipid metabolism, we investigated the effect of PID1 on the content and biological activities of serum lipoproteins in pigs. PID1-transgenic pigs were genetated by sperm and magnetic nanoparticles-mediated method. The levels of PID1 in PID1-transgenic pig's liver were higher than that in the wild-type pig's liver. We found that serum levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (apoA-I) were significantly reduced in PID1-transgenic pigs. On the other hand, PID1-transgenic pigs displayed increased non-HDL-C levels. Serum levels of total cholesterol and triglycerides were comparable between the PID1-transgenic and the wild-type pigs. Further, the HDL isolated from PID1-transgenic pigs showed a significant reduction in cholesterol efflux ability. In addition, serum superoxide dismutase activity of PID1-transgenic pigs was also obviously lowered compared with that of wild type pigs. In conclusion, these results suggest that PID1 might be able to adjust HDL-C levels in serum and HDL cholesterol efflux ability.

Osteopontin Promotes Colorectal Cancer Cell Invasion and the Stem Cell-Like Properties through the PI3K-AKT-GSK/3ß-ß/Catenin Pathway.

BACKGROUND Osteopontin (OPN) is a molecule expressed in numerous cancers including colorectal cancer (CRC) that correlates disease progression. The interaction of OPN that promotes CRC cell migration, invasion, and cancer stem-like cells (CSCs) have not been elucidated. Hence, we aimed to investigate the mechanisms that might be involved. MATERIAL AND METHODS Expression of OPN in tumor tissues derived from patients was monitored with real-time quantitative polymerase chain reaction and western blot. Wound healing and Transwell assay were used to test the differences in migration and invasion in an OPN enriched environment and OPN knockdown condition. Aldehyde dehydrogenase 1 (ALDH1) positive stem cells were isolated using fluorescence-activated cell sorting (FACS) following the protocol of the ALDEFLUOR™ kit. The expression of protein participation in the PI3K-Akt-GSK/3ß-ß/catenin pathway was detected by western blot. RESULTS OPN exhibited increased levels in CRC tumor tissue compared with non-tumor normal tissue and the high level of which correlated with lymphatic metastasis and late TNM stage. Additional rhOPN co-cultured low-expression CRC cells demonstrated more aggressive capability of proliferation, migration, and invasion. For knockdown of OPN in high-expression CRC cells, the bioactivities of proliferation, migration, and invasion were significantly inhibited. Interestingly, the percentage of ALDH1 labeled stem cells was dramatically decreased by OPN inhibition. The phosphorylation of PI3K-Akt-GSK/3ß-ß/catenin pathway was involved in the OPN signaling. Furthermore, Ly294002, a specific PI3K inhibitor, can reverse the promotion of bioactivities and stem cell proportion among rhOPN treated CRC cells. CONCLUSIONS OPN promoted cell proliferation, migration, and invasion, and was accompanied by upregulation of ALDH1-positive CSC in CRC through activation of PI3K-Akt-GSK/3ß-ß/catenin pathway.

Study on the failure characteristics of sliding surface and stability analysis of inverted t-type retaining wall in active limit state.

This paper investigates the sliding surface failure characteristics, earth pressure distribution law and stability safety factor of inverted T-type retaining wall by using the finite element limit analysis software OptumG2, the effects of width of wall heel plate, width of wall toe plate, thickness of bottom plate, soil-wall interface friction angle, soil cohesion and soil internal friction angle of filling on the failure characteristics of sliding surface, the earth pressure distribution law and stability safety factor of retaining walls are analyzed, The stability safety factor of the retaining wall showed a gradually increasing trend as the width of wall heel plate and wall toe plate increased; as the bottom plate thickness increases, the stability safety factor of the retaining wall gradually increases; as the soil-wall interface element reduction coefficient rises, that is, the internal friction angle of the soil-wall gradually increases to the soil internal friction angle, the stability safety factor of the retaining wall gradually increases; as the soil cohesion and internal friction angle increase, the stability safety factor of the retaining wall progressively increases. The safety factor of retaining wall increases by 0.45 for every 0.5m increase in the width of the wall heel plate; the safety factor of the retaining wall increases by 0.29 when the width of the wall toe plate increases by 0.5m; for every 0.5m increase in the width of wall plate thickness, the safety factor of the retaining wall is increased by 0.62; for every 0.25 increase in soil-wall interface element reduction coefficient, the safety factor of the retaining wall increases by 0.29; for every increase of 5KPa in soil cohesion, the safety factor of the retaining wall increased by 1.16; for every 5° increases in soil internal friction angle, the safety factor of retaining wall increases by 0.6. The research is significant for studying the failure laws and stability of retaining walls and providing references for retaining wall design.

Identification and functional prediction of long non-coding RNAs related to oxidative stress in the jejunum of piglets.

OBJECTIVE: Oxidative stress (OS) is a pathological process arising from the excessive production of free radicals in the body. It has the potential to alter animal gene expression and cause damage to the jejunum. However, there have been few reports of changes in the expression of long noncoding RNAs (lncRNAs) in the jejunum in piglets under OS. The purpose of this research was to examine how lncRNAs in piglet jejunum change under OS. METHODS: The abdominal cavities of piglets were injected with diquat (DQ) to produce OS. Raw reads were downloaded from the SRA database. RNA-seq was utilized to study the expression of lncRNAs in piglets under OS. Additionally, six randomly selected lncRNAs were verified using quantitative real-time polymerase chain reaction (qRT‒PCR) to examine the mechanism of oxidative damage. RESULTS: A total of 79 lncRNAs were differentially expressed (DE) in the treatment group compared to the negative control group. The target genes of DE lncRNAs were enriched in gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways. Chemical carcinogenesis-reactive oxygen species, the Foxo signaling pathway, colorectal cancer, and the AMPK signaling pathway were all linked to OS. CONCLUSION: Our results demonstrated that DQ-induced OS causes differential expression of lncRNAs, laying the groundwork for future research into the processes involved in the jejunum's response to OS.

A case study on soil slope landslide failure and parameter analysis of influencing factors for safety factor based on strength reduction method and orthogonal experimental design.

In civil engineering, stability analysis of slope is one of the main content of design. By using the finite element limit analysis software OptumG2, a landslide geological model is established to simulate the failure process of the landslide in Huadu District, Guangzhou City, China. The analysis focused on the deformation and failure characteristics, as well as the mechanical mechanism of landslide; the landslide mode of homogeneous soil is circular sliding. Additionally, investigating the influencing factors affecting slope stability is crucial in engineering implementation; in which the five influencing factors are considered as follow: slope height, slope gradient, soil cohesion, soil internal friction angle, and soil unit weight, respectively. A stability calculation model for a soil slope is established under 25 working conditions based on strength reduction method and orthogonal experimental design, in which the relationship between the safety factor and slope height, slope gradient, soil cohesion, soil internal friction angle, and soil unit weight is obtained. As the slope height increases from 5m to 45m, the safety factor of soil slope gradually decreases from 2.21 to 0.94; As the slope gradient increases from 20° to 60°, the safety factor of soil slope decreases approximately linearly from 1.80 to 0.95; As the cohesion of soil increases from 10kpa to 30kpa, the safety factor of soil slope increases approximately linearly from 1.04 to 1.60; As the internal friction angle of soil increases from 10° to 30°, the safety factor of soil slope increases approximately linearly from 1.00 to 1.81; As the unit weight of soil increases from 13kN/m3 to 21kN/m3, the safety factor of soil slope decreases approximately linearly from 1.50 to 1.21. The influencing factors affecting the safety factor of soil slope in descending order are slope height, slope angle, soil internal friction angle, soil cohesion, and soil unit weight. The research has reference significance for studying the stability and failure laws of soil slopes and conducting landslide control on soil slopes.

Molecular characterization and expression analysis of NDUFS4 gene in m. longissimus dorsi of Laiwu pig (Sus scrofa).

To study the molecular basis of intramuscular fat (IMF) deposition, suppression subtractive hybridization was used to investigate the differences in gene expression between m. longissimus dorsi (LD) of high IMF Laiwu pig group and low IMF Laiwu pig group. From two specific subtractive cDNA libraries, the expression-upregulated clone HL-27 was selected by reverse Northern high-density blot, and then identified to be pig mitochondrial NADH dehydrogenase (ubiquinone) Fe-S protein 4 (NDUFS4). Pig NDUFS4 full-length cDNA was cloned by RACE, and contains a 528 bp-open reading frame (ORF) encoding 175 amino acid residues. The derived amino acid sequence of NDUFS4 is well conserved compared with NDUFS4 of various species with higher degree of sequence similarity with other mammalian (86.3-92.6 %) than amphibian, aves, and fishes (70.2-81.1 %), and contains one N-linked glycosylation site, one O-linked glycosylation site, seven Ser phosphorylation sites and five Thr phosphorylation sites. A-G mutation was found at nt 122 site of ORF between Laiwu pig and Large White, which results in the K-R mutation at 41 site of protein sequence. Real-time PCR analysis indicated that the level of NDUFS4 mRNA expression was higher in high IMF Laiwu pig group than in low IMF Laiwu pig group, and in Laiwu pig than in Large White. The tissue expression of the pig NDUFS4 gene showed a tissue-specific pattern: highly expressed in LD muscle, spleen and kidney, but hardly expressed in lung, stomach and large intestine. The possible role of NDUFS4 and its relation to IMF deposition are discussed.

Analysis on differential expressed genes of ovarian tissue between high- and low-yield laying hen.

In order to elucidate molecular genetic mechanism of laying hen reproduction at the transcriptional level and the structure of significantly differential genes, the mRNA differential display and reverse northern dot-blot were used to detect the differential expression of genes in the ovary tissue of low-yield laying hens and high-yield laying hens in the present study. Sixteen 32-week-old CAU-pink laying hens divided into two groups were used and the laying performance was measured. The results showed that only the egg numbers were significantly different between the two groups; and from 15 primer pairs, a total of 336 bands were displayed of which 59 cDNA bands were found to be differentially expressed in both high-yield and low-yield laying hen. The sequence analysis indicated that the expression of such bands as H-AP5, H-P5, and H-P4 was significantly potentiated in high-yield laying hen using primer pairs AP5/HT11G, P5/HT11G and P4/HT11G and these transcripts had high homology (98%) to HoxDb, HoxCa, and HoxBa, respectively. The differentially expressed gene fragments may be relevant to the progression of the high-yield hens to the egg-laying stage. And further study is required to elucidate the molecular function to improve the productivity of laying hens.

miR-26a-5p Regulates Adipocyte Differentiation via Directly Targeting ACSL3 in Adipocytes.

Preadipocytes become mature adipocytes after proliferation and differentiation, and although many genes and microRNAs have been identified in intramuscular fat, their physiological function and regulatory mechanisms remain largely unexplored. miR-26a-5p has been reported to be related to fat deposition, but its effect on porcine preadipocyte differentiation has not been explored. In this study, bioinformatics analysis and luciferase reporter assay identified that miR-26a-5p binds to the 3'UTR of Acyl-CoA synthetase long-chain family member 3 (ACSL3) mRNA. The model for porcine intramuscular preadipocyte differentiation was established to explore the function of miR-6a-5p-ACSL3 on adipocyte differentiation. ACSL3 knockdown markedly reduced the triglycerides (TG) content of cells, as well as the mRNA levels of adipogenic marker genes (PPAR-γ and SREBP-1c). The number of lipid droplets in cells transfected with a miR-26a-5p mimic is significantly reduced, consistent with ACSL3 knockdown results, while the miR-26a-5p inhibitor resulted in opposite results. Taken together, miR-26a-5p is a repressor of porcine preadipocyte differentiation and plays a vital role in ACSL3-mediated adipogenesis.

Rheo-Fermentation Dough Properties, Bread-Making Quality and Aroma Characteristics of Red Bean (Vigna angularis) Sourdough Induced by LAB Weissella confusa QS813 Strain Fermentation.

This study investigated the impact of in situ-formed exopolysaccharides (EPS) in red bean (Vigna angularis) sourdough fermented by Weissella confusa QS813 on dough rheo-fermentation properties, bread-making quality and aroma characteristics of red bean sourdough bread. The EPS formed in red bean sourdough and sourdough-induced acidification improved the maximum dough fermentation height, gas retention coefficient and viscoelastic properties of dough. Doughs had a lower increase rate of total SDS-soluble gluten proteins, a low decline in GMP content and similar free sulfhydryl content to wheat dough. Resultantly, breads showed declines in baking loss and hardness, increase in specific volume and lower moisture loss and staling rate after 7 days of storage. Finally, despite a reduction in the total content of aroma compounds, new aroma compounds such as acetic acid and higher contents of 3-methyl-1-butanol and 2,3-butanediol were enriched in red bean sourdough bread. Sourdough acidification probably promoted interaction of EPS with gluten or red bean proteins through bond interactions to form structures which stabilized gluten in dough and increased water-binding ability in red bean sourdough bread. This study provided a better understanding of the role of EPS in sourdough in improving bread quality and of promising strategies to address consumer demand for nutritious and clean-label products.

Characteristics of the microstructure and the key components of white kidney bean sourdough bread induced by mixed-strain fermentation and its influence on gut microbiota.

In this study, the effect of mixed-strain fermentation using Kluyveromyces marxianus with either Lactobacillus plantarum or Pediococcus pentosaceus on the physiochemical and nutritional properties of white kidney bean flour sourdough was investigated. The results indicated that mixed-strain fermentation reduced the anti-nutritional factors produced from the white kidney bean flour, especially in the sourdough fermented by L. plantarum and K. marxianus (WKS-LK) compared to that by P. pentosaceus and K. marxianus (WKS-JK). Meanwhile, the content of lactic acid and acetic acid and the proportion of peptides with molecular weights ranging from <500 to 5000 Da were increased in the sourdoughs (WKS-LK > WKS-JK). Compared to the control (WK), microstructural characteristics of the dough seemed to be improved in WKS-LK followed by WKS-JK in terms of their corresponding gluten network consistency. Moreover, mixed fermentation led to a reduced starch digestibility accompanied by a higher content of resistant starch and slowly digestible starch. In contrast, protein digestibility was enhanced in WKS-LK and WKS-JK sourdough breads. More importantly, the changes in gut microbiota composition, short-chain fatty acid (SCFA) production, systemic inflammation, glucose tolerance and liver tissue histopathology following 21-day consumption of the sourdough bread were also evaluated via an animal model. The intake of sourdough breads reduced the abundance of the pathogenic microbiota Escherichia shigella. In contrast, the corresponding abundance of Rikenellaceae, Akkermansiaceae, Erysipelotrichaceae, Prevotellaceae and Eubacterium coprostanoligenes was increased, followed by enhanced SCFA generation, with the highest in WKS-LK and then WKS-JK. Meanwhile, a reduced level of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α in the serum and improved glucose tolerance and liver tissue histopathology following the bread consumption were also achieved in the order of WKS-LK, then WKS-JK mice compared to WK.

Pangenome obtained by long-read sequencing of 11 genomes reveal hidden functional structural variants in pigs.

Long-read sequencing (LRS) facilitates both the genome assembly and the discovery of structural variants (SVs). Here, we built a graph-based pig pangenome by incorporating 11 LRS genomes with an average of 94.01% BUSCO completeness score, revealing 206-Mb novel sequences. We discovered 183,352 nonredundant SVs (63% novel), representing 12.12% of the reference genome. By genotyping SVs in an additional 196 short-read sequencing samples, we identified thousands of population stratified SVs. Particularly, we detected 7,568 Tibetan specific SVs, some of which demonstrate significant population differentiation between Tibetan and low-altitude pigs, which might be associated with the high-altitude hypoxia adaptation in Tibetan pigs. Further integrating functional genomic data, the most promising candidate genes within the SVs that might contribute to the high-altitude hypoxia adaptation were discovered. Overall, our study generates a benchmark pangenome resource for illustrating the important roles of SVs in adaptive evolution, domestication, and genetic improvement of agronomic traits in pigs.

Molecular cloning and expression analysis of tyrosinase gene in the skin of Jining Gray Goat (Capra hircus).

Tyrosinase is the key regulatory enzyme of melanogenesis and plays a major role in mammal coat color. For the first time, we have sequenced and characterized the tyrosinase (TYR) of Jining Gray Goat (Capra hircus), which is the world-famous fur-bearing animal with its special color and pattern. The full-length cDNA was cloned by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method. As a result, one 2131-bp nucleotide sequence representing the full-length cDNA of TYR was obtained. The entire open reading frame (ORF) of the TYR is 1593 bp and encodes for 530 amino acids, which is well conserved compared with TYR of various species with higher degree of sequence similarity with other mammalian (74-99 %) than amphibian, aves, and fishes (56-73 %). The deduced amino acids contained one signal peptide, one transmembrane domain, five N-linked glycosylation sites, and two copper binding sites. The result of real-time quantitative PCR showed that the expression level of TYR was the highest in the dark-gray goats and the lowest in the light-gray ones, while the goats of dark-gray individuals have more than 50 % black fiber and light-gray ones less than 30 %. During the whole life of Jining gray goat, TYR expression level changes with certain regularity and their coat color will change correspondingly by investigating the expression level in ten development stages. After comparing the result and the coat phenotype, we presume that it seems to have a positive relationship between the color depth of coat and the expression level of TYR.

Molecular cloning, characterization and expression of cathepsin D from grass carp (Ctenopharyngodon idella).

Cathepsin D is a lysosomal aspartic proteinase which participates in various degradation functions within the cell. In this current study, we cloned and characterized the complete cDNA of grass carp cathepsin D through 5'- and 3'-RACE. The cathepsin D contained a 56 bp 5' terminal untranslated region (5'-UTR), a 1197 bp open reading frame encoding 398 amino acids, and a 394 bp 3'-UTR. Grass carp cathepsin D shared high similarity with those from other species, and showed the highest amino acid identity of 91% to Danio rerio. Unlike many other organisms, the grass carp cathepsin D contains only one N-glycosylation site closest to the N-terminal. Real-time quantitative RT-PCR demonstrated that Cathepsin D expressed in all twelve tissues (bladder, brain, liver, heart, gill, muscle, fin, eye, intestines, spleen, gonad and head kidney). The relative expression levels of Cathepsin D in gonad and liver were 26.58 and 24.95 times as much as those in fin, respectively. The expression level of Cathepsin D in muscle approximately 16-fold higher, in intestines and spleen were 12-fold higher. The cathepsin D expression showed an upward trend during embryonic development. After challenged with Aeromonas hydrophil, the expression of grass carp cathepsin D gene showed significant changes in the four test tissues (liver, head kidney, spleen and intestines). The fact that the bacterial infection can obviously improve the cathepsin D expression in immune-related organs, may suggest that cathepsin D plays an important role in the innate immune response of grass carp.

Establishment and characterization of outer root sheath (ORS) cell line from Jining grey goat.

A new line of outer root sheath (ORS) cells was established from hair follicles of Jining grey goat by using a mechanical separation combined with enzyme digestion. Cell morphology is described at different phases. The chromosome analysis of ORS cells, identification of the ORS cells and morphological reversion test were detected at the 4th and 40th passages. The ORS cells were healthy and the growth characteristics were stable with a population doubling time of 52 h. Chromosome analysis showed that >58% of cells were diploid. Test for ORS cell line CK19 expression was positive. This newly established ORS cell line not only lays the foundation for further studying on the growth, regeneration, development law of goat hair follicle but also provides a mirror for the research of human hair in medical field.

Comparative Analysis on Antioxidative Ability of Muscle between Laiwu Pig and Large White.

This study was conducted to evaluate effects of storage temperatures (4°C and 20°C) and pig breeds (Laiwu pig and Large White pig) on the main antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidase) activity and lipid oxidation in porcine Longissimus dorsi muscle. Activities of antioxidative enzymes (AOE) decreased slightly during storage, regardless of storage temperatures. Muscle antioxidative enzymes activities stored at 4°C were higher than that stored at 20°C. Laiwu pig's enzymes activities were significantly (p<0.01) higher than Large White's. The level of malondialdehyde is a direct expression of the grade of lipid oxidation in meat. In our study, the malondialdehyde contents increased after 6 days storage. However, malondialdehyde contents of Laiwu pig were significantly (p<0.01) lower than Large White's. A lower content of malondialdehyde corresponds to a lower oxidation of lipids. These results indicated the muscle antioxidative ability of Laiwu pig was higher than Large White pig. It also implied that antioxidative enzymes were involved in the essentials and deciding mechanisms of meat quality by quenching oxygen free radicals and inhibiting lipid oxidation in muscle.