Production of cleavage-resistant phytase transgenic pigs by handmade cloning.

With the rapid development of intensive animal husbandry in the livestock industry, large quantities of manure waste containing phytate phosphorus are being generated. Phytase can effectively solve the problem of high phosphorus pollution in the feces of monogastric animals. Enviropig, which produces phytase in the salivary glands and secretes the enzyme in the saliva, were first generated in 1999. However, phytase is easily inactivated during digestion. To address this problem, cleavage-resistant phytase transgenic pigs were generated using handmade cloning in this study. Transgene construction was improved and three cell lines carrying Cafp were obtained. In total, 810 blastocysts were generated and 712 good-quality were transferred into six recipients. Fourteen piglets were born, of which six survived after weaning. Polymerase chain reaction and sequencing results showed that seven (three live and four dead) of the fourteen piglets carried Cafp. Phytase activity in the saliva of the six live cloned pigs was tested at four months of age, and only one pig had 0.155 FTU/mL enzyme activity. The other five pigs may not have been activated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, representing a 25.4% decrease in fecal emission compared to the average of controls. Immunohistochemical results on the three Cafp-positive pigs that died after six months of age showed that the transgene was only expressed in parotid glands, confirming tissue-specific gene expression. In conclusion, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to managing phosphorus nutrition and environmental pollution in animal husbandry.

Leptin Receptor Gln223Arg Polymorphism of Human Spermatozoa Associated with Male Infertility in a Chinese Population.

Background: Leptin (LEP) is believed to play a crucial role in male reproduction, while the molecular mechanisms through which LEP affects the male reproductive system are unclear. LEP acts by binding to a leptin receptor (LEPR) which mediates its physiological action, but there are only limited studies on the function of LEPR in human sperm. Purpose: This study aimed to determine the Gln223Arg polymorphisms of the LEPR gene in human spermatozoa and evaluate their possible relationship with semen variables. Methods: The study was performed on Chinese men: 115 healthy subjects and 108 patients with primary and 98 with secondary infertility. Semen samples were obtained from all patients, and semen variables were analyzed. The genotypic and allelic frequencies of Gln223Arg polymorphism in spermatozoa were determined by PCR and restriction fragment length polymorphism (RFLP) analyses. Statistical analyses were performed using the chi-square test, the Kruskal-Wallis test, and the Mann-Whitney test. Results: There were no significant differences in genotypic or allelic frequency distributions of Gln223Arg polymorphism among men with primary infertility, secondary infertility, and controls. Similarly, semen volume and sperm concentration did not differ with the different genotypes in all groups of men. The percentages of motile sperm for AA + AG genotypes in men with primary infertility (31.98%) were significantly lower than those in secondary infertility, and control men with GG genotypes were 34.41% and 59.36%, respectively. At the same time, the percentages of normal morphology sperm for AA + AG genotypes in men with primary infertility (2.93%) were significantly lower than those in secondary infertility and control men with GG genotypes 3.71% and 6.54%, respectively. Conclusion: This study reveals a possible association between the Gln223Arg polymorphism of the LEPR gene in spermatozoa affecting spermatozoal membrane integrity and having a direct role in sperm motility.

Negative correlation between acetyl-CoA acyltransferase 2 and cetuximab resistance in colorectal cancer.

The emergence of anti-EGFR therapy has revolutionized the treatment of colorectal cancer (CRC). However, not all patients respond consistently well. Therefore, it is imperative to conduct further research to identify the molecular mechanisms underlying the development of cetuximab resistance in CRC. In this study, we find that the expressions of many metabolism-related genes are downregulated in cetuximab-resistant CRC cells compared to their sensitive counterparts. Specifically, acetyl-CoA acyltransferase 2 (ACAA2), a key enzyme in fatty acid metabolism, is downregulated during the development of cetuximab resistance. Silencing of ACAA2 promotes proliferation and increases cetuximab tolerance in CRC cells, while overexpression of ACAA2 exerts the opposite effect. RTK-Kras signaling might contribute to the downregulation of ACAA2 expression in CRC, and ACAA2 predicts CRC prognosis in patients with Kras mutations. Collectively, our data suggest that modulating ACAA2 expression contributes to secondary cetuximab resistance in Kras wild-type CRC patients. ACAA2 expression is related to Kras mutation and demonstrates a prognostic role in CRC patients with Kras mutation. Thus, ACAA2 is a potential target in CRC with Kras mutation.

JUNB mediates oxaliplatin resistance via the MAPK signaling pathway in gastric cancer by chromatin accessibility and transcriptomic analysis.

Currently, platinum-containing regimens are the most commonly used regimens for advanced gastric cancer patients, and chemotherapy resistance is one of the main reasons for treatment failure. Thus, it is important to reveal the mechanism of oxaliplatin resistance and to seek effective intervention strategies to improve chemotherapy sensitivity, thereby improving the survival and prognosis of gastric cancer patients. To understand the molecular mechanisms of oxaliplatin resistance, we generate an oxaliplatin-resistant gastric cancer cell line and conduct assay for transposase-accessible chromatin sequencing (ATAC-seq) and RNA sequencing (RNA-seq) for both parental and oxaliplatin-resistant AGS cells. A total of 3232 genomic regions are identified to have higher accessibility in oxaliplatin-resistant cells, and DNA-binding motif analysis identifies JUNB as the core transcription factor in the regulatory network. JUNB is overexpressed in oxaliplatin-resistant gastric cancer cells, and its upregulation is associated with poor prognosis in gastric cancer patients, which is validated by our tissue microarray data. Moreover, chromatin immunoprecipitation sequencing (ChIP-seq) analysis reveals that JUNB binds to the transcriptional start site of key genes involved in the MAPK signaling pathway. Knockdown of JUNB inhibits the MAPK signaling pathway and restores sensitivity to oxaliplatin. Combined treatment with the ERK inhibitor piperlongumine or MEK inhibitor trametinib effectively overcomes oxaliplatin resistance. This study provides evidence that JUNB mediates oxaliplatin resistance in gastric cancer by activating the MAPK pathway. The combination of MAPK inhibitors with oxaliplatin overcomes resistance to oxaliplatin, providing a promising treatment opportunity for oxaliplatin-resistant gastric cancer patients.

PROX1-mediated epigenetic silencing of SIRT3 contributes to proliferation and glucose metabolism in colorectal cancer.

Prospero-related homeobox 1 (PROX1) is a homeobox transcription factor known to promote malignant transformation and stemness in human colorectal cancer (CRC). However, the biological function of PROX1 in metabolic rearrangement in CRC remains unclear. Here, we aimed to uncover the relationship between the expression profile and role of PROX1 and CRC cell glucose metabolism and to elucidate the underlying molecular mechanism. PROX1 expression was significantly upregulated in human CRC tissues and positively associated with the maximum standardized uptake value (SUVmax), a measure of tissue 18-fluoro-2-deoxy-D-glucose uptake and an indicator of glycolysis and tumor cell activity, in patients with CRC. Knockdown of PROX1 suppressed CRC cell proliferation and glucose metabolism in vitro and in vivo. Mechanistically, through a physical interaction, PROX1 recruited EZH2 to the SIRT3 promoter and inhibited SIRT3 promoter activity. Moreover, PROX1 or EZH2 knockdown decreased cell glycolysis by targeting SIRT3. Clinically, high PROX1 expression combined with low SIRT3 expression predicted poor prognosis in patients with CRC. Thus, our study suggests that the PROX1-EZH2 complex positively regulates cell proliferation and glucose metabolism by engaging SIRT3 in CRC, which may serve as a promising therapeutic strategy for CRC.

Leptin Levels in Serum or Semen and Its Association with Male Infertility: A Meta-Analysis with 1138 Cases.

Background: Leptin has an association with male infertility. However, only sporadic studies inconsistently reported the results. Aim and Objective. In this study, we aimed to perform a meta-analysis to investigate the relationship between leptin and male infertility. Methods: This study was performed based on published articles related to leptin and infertile males. PubMed, Web of Science, Google Scholar, Ovid + Cochrane Central Register of Controlled Trials, Wiley Online Library, Chinese CNKI, Chinese Chong Qing VIP, Chinese Wan Fang, and China Biology Medicine databases were searched to identify all relevant studies. All eligible works of literature were analyzed by the "meta" or "metan" command in STATA version 12.0 software. The standardized mean difference (SMD) of leptin concentration in serum or semen and 95% confidence intervals (CIs) were estimated for all studies. The heterogeneity was described with I2. The sources of heterogeneity were explored via metaregression, and stratified analyses, sensitivity analyses, and publication bias were performed. Results: Nineteen studies were included in the current meta-analysis, involving 1138 cases of infertile men and 756 controls. The SMD of leptin concentration in serum was 2.002 (95% CI: 1.086, 2.918), Z-test (z) z = 4.29; p < 0.001, and I2 was 97.3%, p < 0.001. The SMD of leptin concentration in semen was 3.274 (95% CI: 2.137, 4.411), z = 5.64; p < 0.001, and I2 was 98.2%, p < 0.001. Notably, serum follicle-stimulating hormone (FSH) was slightly higher in infertile men (SMD = 3.695, z = 2.33, p = 0.020, I2 = 98.8%, p < 0.001). Other hormones, such as luteinizing hormone (LH) and testosterone, were also slightly higher, but the results were not statistically significant. In addition, sperm count (SMD = -4.533, 95% CI: -6.565, -2.501) and sperm motility (SMD = -7.894, 95% CI: -10.616, -5.172) inversely correlated with leptin levels in infertile males. Sperm abnormal forms did not show a statistically significant SMD of -0.076 (95% CI: -3.410, 3.258). Conclusion: Leptin plays a potential role in association with male infertility. This study may effectively reveal the relationship between leptin together with other hormones and its association with male infertility. These results may also provide opinions on precautionary measures.

Molecular signatures of tumor progression in pancreatic adenocarcinoma identified by energy metabolism characteristics.

BACKGROUND: In this study, we performed a molecular evaluation of primary pancreatic adenocarcinoma (PAAD) based on the comprehensive analysis of energy metabolism-related gene (EMRG) expression profiles. METHODS: Molecular subtypes were identified by nonnegative matrix clustering of 565 EMRGs. An overall survival (OS) predictive gene signature was developed and internally and externally validated based on three online PAAD datasets. Hub genes were identified in molecular subtypes by weighted gene correlation network analysis (WGCNA) coexpression algorithm analysis and considered as prognostic genes. LASSO cox regression was conducted to establish a robust prognostic gene model, a four-gene signature, which performed better in survival prediction than four previously reported models. In addition, a novel nomogram constructed by combining clinical features and the 4-gene signature showed high-confidence clinical utility. According to gene set enrichment analysis (GSEA), gene sets related to the high-risk group participate in the neuroactive ligand receptor interaction pathway. CONCLUSIONS: In summary, EMRG-based molecular subtypes and prognostic gene models may provide a novel research direction for patient stratification and trials of targeted therapies.

Prognostic Factors and a Nomogram Predicting Survival in Patients with Breast Ductal Carcinoma in situ with Microinvasion: A Population-Based Study.

PURPOSE: Ductal carcinoma in situ with microinvasion (DCISM) can be challenging to balance the risks of overtreatment versus undertreatment. We aim to identify prognostic factors in patients with DCISM and construct a nomogram to predict breast cancer-specific survival (BCSS). MATERIALS AND METHODS: A retrospective cohort study of women diagnosed with DCISM from 1988 to 2015 who were identified in the Surveillance, Epidemiology and End Results database. Clinical variables and tumor characteristics were evaluated, and Cox proportional-hazards regression was performed. A nomogram was constructed from the multivariate logistic regression to combine all the prognostic factors to predict the prognosis of DCISM patients at 5 years, 10 years, and 15 years. RESULTS: We identified 5438 total eligible breast cancer patients with a median and max survival time of 78 and 227 months, respectively. Here, patients with poorer survival outcomes were those diagnosed between 1988 and 2001, African-American race, under 40 years of age, higher tumor N stage, progesterone receptor-negative tumor, and received no surgery. The nomogram was constructed by the seven variables and passed the calibration and validation steps. The area under the receiver operating characteristic (ROC) curve (AUC) of both the training set and the validating set (5-year AUC: 0.77 and 0.88, 10-year AUC: 0.75 and 0.73, 15-year AUC: 0.72 and 0.65). Receiving chemotherapy was associated with a better BCSS (hazard ratio, HR=0.45, 95% confidence interval, 95% CI = 0.23-0.89), especially in patients with estrogen receptor (ER) negative, progesterone receptor (PR) negative (HR = 0.35, 95% CI = 0.13-0.97) and ER+PR-/ER-PR+ DCISM (HR = 0.07, 95% CI = 0.01-0.59). CONCLUSION: Our current study is the first to construct nomograms of patients with DCISM which could help physicians identify breast cancer patients that more likely to benefit from more intensive treatment and follow-up. Chemotherapy might benefit patients with ER-PR- and ER+PR-/ER-PR+ DCISM.

Transcriptomic analysis of tumor tissues and organoids reveals the crucial genes regulating the proliferation of lung adenocarcinoma.

BACKGROUND: Accumulative evidence shows that an organoid is a more practical and reliable tool in cancer biology research. This study aimed to identify and validate crucial genes involved in non-small cell lung cancer carcinogenesis and development using the transcriptomic analysis of tumor tissues and organoids. METHODS: Gene set enrichment analysis (GSEA) of tumor tissues, tumor organoids, and normal tissues was performed to reveal the similar and different mechanisms involved in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) carcinogenesis and progression. Differentially expressed gene analysis, prognostic analysis, and gene co-expression network analysis were further used to identify hub genes involved in LUAD and LUSC carcinogenesis and development. Finally, LUAD cell lines and organoids were used to validate these findings. RESULTS: GSEA analysis was performed to reveal the similar mechanisms involved in LUAD and LUSC carcinogenesis and development, such as P53 signaling pathway, base mismatch repair, DNA replication, cAMP signaling pathway and PPAR pathway. However, comparing with LUSC organoids, LUAD organoids showed downregulation of immune-related pathways, inflammation-related pathways, MAPK signaling pathways, and Rap1 signaling pathways, although these pathways were downregulated in LUAD and LUSC tissues by comparing with normal lung tissues. Further gene co-expression network analysis and prognostic analysis indicated CDK1, CCNB2, and CDC25A as the hub tumor-promoting genes in LUAD but not in LUSC, which were further validated in other datasets. Using LUAD cell lines and organoid models, CDK1 and CCNB2 knockdown were found to suppress LUAD proliferation. However, CDC25A knockdown did not inhibit LUAD cell line proliferation but could effectively suppress LUAD organoid growth, indicating that an organoid could be used as an effective tool to study cancer biology in LUAD. CONCLUSIONS: The results revealed CDK1, CCNB2, and CDC25A as the hub genes involved in LUAD carcinogenesis and development, which could be used as the potential biomarkers and targets for LUAD.

Hepatopancreatic metabolomics shedding light on the mechanism underlying unsynchronized growth in giant freshwater prawn, Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii (M. rosenbergii) as an important freshwater aquaculture species with high commercial value, exhibited unsynchronized growth. However, the potentially metabolic mechanism remains unclear. In this study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the hepatopancreatic metabolic profiles of twenty giant freshwater prawns between the fast-growing group and slow-growing group. In the metabolomics assay, we isolated 8,293 peaks in positive and negative iron mode. Subsequently, 44 significantly differential metabolites were identified. Functional pathway analysis revealed that these metabolites were significantly enriched in three key metabolic pathways. Further integrated analysis indicated that glycerophospholipid metabolism and aminoacyl-tRNA biosynthesis have significant impact on growth performance in M.rosenbergii. Our findings presented here demonstrated the critical metabolites and metabolic pathways involved in growth performance, moreover provided strong evidence for elucidating the potentially metabolic mechanism of the unsynchronized growth in M. rosenbergii.

Analysis of Population Structure and Differentially Selected Regions in Guangxi Native Breeds by Restriction Site Associated with DNA Sequencing.

Guangxi indigenous chicken breeds play a very important role in promoting the high-quality development of the broiler industry in China. However, studies on genomic information of Guangxi indigenous chicken to date remain poorly explored. To decipher the population genetic structure and differentially selected regions (DSRs) in Guangxi indigenous chickens, we dug into numerous SNPs from seven Guangxi native chickens (GX) by employing the restriction site associated with DNA sequencing (RAD-seq) technology. Another three breeds, Cobb, White Leghorn, and Chahua (CH) chicken, were used as a control. After quality control, a total of 185,117 autosomal SNPs were kept for further analysis. The results showed a significant difference in population structure, and the control breeds were distinctly separate from the Guangxi native breeds, which was also strongly supported by the phylogenetic tree. Distribution of FST indicated that there were three SNPs with big genetic differentiation (FST value all reach to 0. 9427) in GX vs. CH group, which were located on chr1-96,859,720,chr4-86,139,601, and chr12-8,128,322, respectively. Besides, we identified 717 DSRs associated with 882 genes in GX vs. Cobb group, 769 DSRs with 476 genes in GX vs. Leghorn group, and 556 DSRs with 779 genes in GX vs. CH group. GO enrichment showed that there were two significant terms, namely GPI-linked ephrin receptor activity and BMP receptor binding, which were enriched in GX vs. Leghorn group. In conclusion, this study suggests that Guangxi native chickens have a great differentiation with Cobb and Leghorn. Our findings would be beneficial to fully evaluate the genomic information on Guangxi native chicken and facilitate the application of these resources in chicken breeding.

Comparative Transcriptome Analysis of Gonads for the Identification of Sex-Related Genes in Giant Freshwater Prawns (MacrobrachiumRosenbergii) Using RNA Sequencing.

: The giant freshwater prawn (Macrobrachiumrosenbergii) exhibits sex dimorphism between the male and female individuals. To date, the molecular mechanism governing gonadal development was unclear, and limited data were available on the gonad transcriptome of M.rosenbergii. Here, we conducted comprehensive gonadal transcriptomic analysis of female (ZW), super female (WW), and male (ZZ) M.rosenbergii for gene discovery. A total of 70.33 gigabases (Gb) of sequences were generated. There were 115,338 unigenes assembled with a mean size of 1,196 base pair (bp) and N50 of 2,195 bp. Alignment against the National Center for Biotechnology Information (NCBI) non-redundant nucleotide/protein sequence database (NR and NT), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, SwissProt database, Protein family (Pfam), Gene ontology (GO), and the eukaryotic orthologous group (KOG) database, 36,282 unigenes were annotated at least in one database. Comparative transcriptome analysis observed that 10,641, 16,903, and 3,393 genes were significantly differentially expressed in ZW vs. ZZ, WW vs. ZZ, and WW vs. ZW samples, respectively. Enrichment analysis of differentially expressed genes (DEGs) resulted in 268, 153, and 42 significantly enriched GO terms, respectively, and a total of 56 significantly enriched KEGG pathways. Additionally, 23 putative sex-related genes, including Gtsf1, IR, HSP21, MRPINK, Mrr, and other potentially promising candidate genes were identified. Moreover, 56,241 simple sequence repeats (SSRs) were identified. Our findings provide a valuable archive for further functional analyses of sex-related genes and future discoveries of underlying molecular mechanisms of gonadal development and sex determination.

Isolation, characterization, and SREBP1 functional analysis of mammary epithelial cell in buffalo.

Compared to cow milk, buffalo milk contains more protein, fat, and vitamin. Buffalo milk is an ideal food in human life. Sterol regulatory element-binding protein 1 (SREBP1), an important transcription factor, regulates the expression and activity of enzyme and protein involved in milk fat synthesis to influence on the synthesis and secretion of triglyceride in mammary epithelial cells. In the present study, we successfully isolated buffalo mammary epithelial cell by using enzymatic digestion, and then described the growth characteristics and expression characteristics of mammary epithelial cells. Moreover, we cloned the SREBP1 gene from total RNA isolated from milk fat globule and analyzed the function of the SREBP1 gene. After infected with shRNA-SREBP1 lentiviral particle and treated with fatty acid, the expression trend of ACACA, FABP3, FAS, SCD, ERK1, ERK2, PPARy, and Insigl genes was consistent with the expression trend of SREBP1 gene. These results suggested that SREBP1 gene is a central transcription factor in regulating milk fat synthesis and SREBP1 gene may act on ERK1/ERK2 signaling pathway to regulate the expression of PPARy gene. The current study will provide a theoretical basis for further reveal the molecular mechanism of milk fat synthesis in buffalo mammary epithelial cells. PRACTICAL APPLICATIONS: This study aim to separate and analysis characterization of mammary epithelial cell in buffalo. Compared to cow milk, buffalo milk contains more protein, fat, and vitamin. Buffalo milk is an ideal food in human life. This study will provide a theoretical basis for further research on the molecular mechanism of milk fat synthesis in buffalo mammary epithelial cells.

Dosage Compensation and Gene Expression of the X Chromosome in Sheep.

Ohno's hypothesis predicts that the expression of the single X chromosome in males needs compensatory upregulation to balance its dosage with that of the diploid autosomes. Additionally, X chromosome inactivation ensures that quadruple expression of the two X chromosomes is avoided in females. These mechanisms have been actively studied in mice and humans but lag behind in domestic species. Using RNA sequencing data, we analyzed the X chromosome upregulation in sheep fetal tissues from day 135 of gestation under control, over or restricted maternal diets (100%, 140% and 60% of National Research Council Total Digestible Nutrients), and in conceptuses, juvenile, and adult somatic tissues. By computing the mean expression ratio of all X-linked genes to all autosomal genes (X:A), we found that all samples displayed some levels of X chromosome upregulation. The degrees of X upregulation were not significant (P-value = 0.74) between ovine females and males in the same somatic tissues. Brain, however, displayed complete X upregulation. Interestingly, the male and female reproduction-related tissues exhibited divergent X dosage upregulation. Moreover, expression upregulation of the X chromosome in fetal tissues was not affected by maternal diets. Maternal nutrition, however, did change expression levels of several X-linked genes, such as sex determination genes SOX3 and NR0B1 In summary, our results showed that X chromosome upregulation occurred in nearly all sheep somatic tissues analyzed, thus support Ohno's hypothesis in a new species. However, the levels of upregulation differed by different subgroups of genes such as those that are house-keeping and "dosage-sensitive".

Characterization and expression of galectin-3 after Streptococcus agalactiae and Aeromonas hydrophila challenge in GIFT strain Nile tilapia (Oreochromis niloticus).

In mammals, Galectin-3 has been revealed to be widely expressed in immune cells and played important role in immune reactions. However, Galectin-3 is frequently less reported in teleost. In the present study, a molecular characterization and expression analysis of galectin-3 were conducted in GIFT strain Nile tilapia. The full-length cDNA is 1034 bp with 690 bp of protein coding sequences. The result of qRT-PCR showed that the mRNA of galectin-3 was widely expressed in various tissues (heart, liver, spleen, gill, kidney, brain, intestine, skin, muscle, and ovary), and the higher expression was observed in immune-related tissues (liver and spleen). The time-course expression analysis revealed that galectin-3 was significantly up-regulated in intestine (5 h, 50 h, and 7 d), liver (5 h, 50 h, and 7 d), spleen (5 and 50 h), head-kidney (5 and 50 h), gill (5 h and 7 d) after Streptococcus agalactiae challenge, and significantly up-regulated in intestine (18, 24, 36, 72, and 96 h), liver (6, 18, 24, 96 h, and 6 d), spleen (18, 24, 36, 72, and 96 h), head-kidney (6, 12, 18, 24, 36, 72, and 96 h), and gill (12, 18, 24, and 36 h) after Aeromonas hydrophila challenge. Taken together, these data suggest that galectin-3 plays a role in immune responses in Nile tilapia after bacterial challenge.

Cryosurvival and pregnancy rates: One-step protocol for freezing-thawing Shangri-la Yak (Bos grunniens) Embryos.

The yak is one of the most important and economically useful animals for highlanders. The decline in the yak population requires effective measures for the conservation and multiplication of elite germplasm. A standardized protocol will simplify the freezing and warming of yak embryos in straw and facilitate embryo transfer. In this work, we investigated a one-step protocol that uses a stable basal medium, which comprised a warming medium (1.08 M sucrose) and a freezing medium (EFS40). We also assessed the effects of the new transfer method on embryo survival. A total of 145 yak frozen embryos were thawed in a standard medium system. The one-step protocol led to a high recovery percentage (84.93) of yak embryos that survived vitrification and warming. The in vitro survival rates of these embryos significantly different from those of embryos frozen-thawed via the conventional method. The 95 embryos frozen-thawed via our one-step protocol were then implanted in selected recipients. Thirty-six singleton pregnancies were established. In conclusion, the proposed one-step method is a simple, safe, and standardized freezing-thawing protocol that ensures embryo survival and quality under field conditions. This study establishes new possibilities for the widespread use of embryo transfer in yaks.

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses.

Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date, 263 imprinted genes have been identified among all investigated mammalian species. However, only 21 have been described in sheep, of which 11 are annotated in the current ovine genome. Here, we aim to i) use DNA/RNA high throughput sequencing to identify new monoallelically expressed and imprinted genes in day 135 ovine fetuses and ii) determine whether maternal diet (100%, 60%, or 140% of National Research Council Total Digestible Nutrients) influences expression of imprinted genes. We also reported strategies to solve technical challenges in the data analysis pipeline. We identified 80 monoallelically expressed, 13 new putative imprinted genes, and five known imprinted genes in sheep using the 263 genes stated above as a guide. Sanger sequencing confirmed allelic expression of seven genes, CASD1, COPG2, DIRAS3, INPP5F, PLAGL1, PPP1R9A, and SLC22A18. Among the 13 putative imprinted genes, five were localized in the known sheep imprinting domains of MEST on chromosome 4, DLK1/GTL2 on chromosome 18 and KCNQ1 on chromosome 21, and three were in a novel sheep imprinted cluster on chromosome 4, known in other species as PEG10/SGCE. The expression of DIRAS3, IGF2, PHLDA2, and SLC22A18 was altered by maternal diet, albeit without allelic expression reversal. Together, our results expanded the list of sheep imprinted genes to 34 and demonstrated that while the expression levels of four imprinted genes were changed by maternal diet, the allelic expression patterns were un-changed for all imprinted genes studied.

Basal polarization of the immune responses to Streptococcus agalactiae susceptible and resistant tilapia (Oreochromis niloticus).

One of the highest priority areas for improvement is the development of effective strategies for decreasing disease mortality levels in aquaculture production, a better understanding of the components of the fish immune system and their functions in the context of pathogen invasion is needed. Tilapia is the most common fish in South China, and Streptococcus agalactiae has become the most serious disease problem for tilapia industry in China. Here, we profiled gene expression differences between tilapia differing in their susceptibility to S. agalactiae both basally (before infection) and at three early timepoints post-infection (5 h, 50 h, and 7 d). Between group comparisons revealed 5756 unique genes differentially expressed greater than 2-fold at one or more timepoints. And the resistant fish showed much more strong ability in pathogen recognition, antigen presentation, immune activation, while the susceptible fish showed fast activation of apoptosis. Taken together, the immune profiles expand our knowledge for molecular mechanisms for disease resistance, as well as provide solid molecular resources for further identification of the candidate markers for disease-resistant selection and evaluation of disease prevention and treatment options for tilapia industry.

Scriptaid Upregulates Expression of Development-Related Genes, Inhibits Apoptosis, and Improves the Development of Somatic Cell Nuclear Transfer Mini-Pig Embryos.

The present study was undertaken to investigate the mechanisms by which Scriptaid treatment improves the developmental competence of somatic cell nuclear transfer (SCNT) mini-pig embryos in vitro. We found that treatment with 500 nmol/L Scriptaid for 15 hours significantly improved the development of mini-pig SCNT embryos. Compared with the control group, the blastocyst rate was higher (18.3% vs. 10.7%; p < 0.05). The acetylation level on H3K14 of the Scriptaid-treated group was higher compared with the control group in SCNT embryos at two-cell, four-cell, and blastocyst stages (p < 0.05). After Scriptaid treatment, histone deacetylase gene HDAC5 expression level was significantly decreased in four-cell embryos and blastocysts, while the expression levels of the embryos' development-related genes AKT, Oct4, and apoptosis inhibited gene PGC-1α were significantly increased in blastocysts (p < 0.05). The number of apoptotic cells per blastocyst in the Scriptaid-treated group was lower compared with the control group (p < 0.05). These results indicate that Scriptaid repressed HDCA5 gene expression, increased the acetylation level of H3K14, upregulated the expression of AKT, Oct4, and PGC-1α genes, improved embryos' development, and reduced apoptosis, which favors development of the SCNT mini-pig embryos to blastocysts.

Transcriptomic profiling analysis of tilapia (Oreochromis niloticus) following Streptococcus agalactiae challenge.

Innate immune system is the primary defense mechanism against pathogen infection in teleost, which are living in pathogen-rich aquatic environment. It has been long hypothesized that the disease resistance in teleost are strongly correlated to the activities of innate immune genes. Tilapia is an important economical fish around the world, especially in China, where the production accounts for nearly half of the global production. Recently, S. agalactiae has become one of the most serious bacterial diseases in southern China, resulted in high cumulative mortality and economic loss to tilapia industry. Therefore, we sought here to characterize the expression profiles of tilapia against S. agalactiae infection at whole transcriptome level by RNA-seq technology. A total of 2822 genes were revealed significantly expressed in tilapia spleen with a general trend of induction. Notably, most of the genes were rapidly the most induced at the early timepoint. The significantly changed genes highlighted the function of pathogen attachment and recognition, antioxidant/apoptosis, cytoskeletal rearrangement, and immune activation. Collectively, the induced expression patterns suggested the strong ability of tilapia to rapidly recognize the invasive bacteria, and activation of downstream immune signaling pathways to clear the bacteria and prevent the tissue damage and bacteria triggered cell apoptosis. Our results heighted important roles of novel candidate genes which were often missed in previous tilapia studies. Further studies are needed to characterize the molecular relationships between key immune genes and disease resistance, and to identify the candidate genes for molecular-assistant selection of disease-resistant broodstock and evaluation of disease prevention and treatment measures.