Search

The early embryonic development starts with the totipotent zygote upon fertilization of differentiated sperm and egg, which undergoes a range of reprogramming and transformation to acquire pluripotency. Induced pluripotent stem cells (iPSCs), a nonclonal technique to produce stem cells, are originated from differentiated somatic cells via accomplishment of cell reprogramming, which shares common reprogramming process with early embryonic development. iPSCs are attractive in recent years due to the potentially significant applications in disease modeling, potential value in genetic improvement of husbandry animal, regenerative medicine, and drug screening. This review focuses on introducing the research advance of both somatic cell reprogramming and early embryonic development, indicating that the mechanisms of iPSCs also shares common features with that of early embryonic development in several aspects, such as germ cell factors, DNA methylation, histone modification, and/or X chromosome inactivation. As iPSCs can successfully avoid ethical concerns that are naturally present in the embryos and/or embryonic stem cells, the practicality of somatic cell reprogramming (iPSCs) could provide an insightful platform to elucidate the mechanisms underlying the early embryonic development.

Different feeding patterns exhibit completely different and heritable growth properties in livestock, but the underlying epigenetic mechanisms remain unclear. Here, we investigated genome-wide DNA-methylation and gene expression under grazing and confinement regimen feeding strategies with Lvliang Black goat. We identified 102 differently expressed genes and 7,833 differentially methylated regions (DMRs) between the two groups. Integrating DNA-methylation and gene expression showed that genes in DMRs exhibit significantly different expression level (FDR < 0.05). KEGG pathway analysis indicated that most of the genes involved in environment adaptation pathways like lipid transpor and metabolism immunity. In sum, our data provided insight into the epigenetic mechanism underlying growth property difference resulting from distinct feeding patterns in goat, and also offered theoretical basis for the rational utilization of germplasm resources of local breeds. Supplemental data for this article is available online at https://doi.org/10.1080/13102818.2021.1914164 .

TGF-β1, an important multi-functional cytokine of the TGF-β signaling pathway, has been reported to be crucial for ovarian granulosa cell (GC) states and female fertility. However, the molecular mechanism underlying TGF-β1 regulation of GC states remains largely unknown. Here, we provide a comprehensive transcriptomic view on TGF-β1 regulation of cell states in porcine GCs. We first confirmed that TGF-β1 can control GC states (apoptosis and proliferation) in pig ovary. RNA-seq showed that 909 differentially expressed genes (DEGs), including 890 DEmRNAs and 19 DEmiRNAs, were identified in TGF-β1-treated porcine GCs. Functional annotation showed that these DEGs were mainly involved in regulating cell states. In addition, multiple hub genes were identified by constructing the protein-protein interaction network, DEmiRNA-DEmRNAs regulatory network, and gene-pathway-function co-expression networks, which were further found to be enriched in FoxO, TGF-β, Wnt, PIK3-Akt, p53 and Ras signaling pathways that play important roles in regulating cell states, cell cycle, proliferation, stress-responses and inflammation. The current research deeply reveals the effects of TGF-β1 on porcine GCs, and also identifies potential therapeutic RNA molecules for inhibiting and rescuing female infertility.

Background Fatty liver disease prevalently occurs in commercial postpartum dairies, resulting in a worldwide high culling rate because of their subsequent limitations of production and reproduction performance. Results Fatty liver-specific proteome and acetylome analysis revealed that energy metabolism suppression closely associated with mitochondrial dysfunction and inflammation activation were shown to be remarkable biological processes underlying the development of fatty liver disease, furthermore, acetylation modification of proteins could be one of the main means to modulate these processes. Twenty pivotal genetic factors/genes that differentially expressing and being acetylation modified in liver were identified and proposed to regulate the pathogenesis of fatty liver dairies. These proteins were confirmed to be differentially expressing in individual liver tissue, eight of which being validated via immunohistochemistry assay. Conclusions This study provided a comprehensive proteome and acetylome profile of fatty liver of dairy cows, and revealed potential important biological processes and essential regulators in the pathogenesis of fatty liver disease. Expectantly, understanding the molecular mechanisms of the pathogenesis of fatty liver disease in dairies, as an animal model of non-alcoholic fatty liver disease (NAFLD) in human beings, which is a clinico-pathologically defined process associated with metabolic syndrome, could inspire and facilitate the development of efficacious therapeutic drugs on NAFLD.

The research direction of our team is nutrition and physiology of ruminants, including dietary nutrition metabolism and rumen microorganisms. Previous research has shown that ruminal acetate-to-propionate ratio is related to diet utilization efficiency. At present, it is believed that the main factors affecting the ruminal acetate-to-propionate ratio are the degradation rate of the diet and the rumen microbial structure, but the main mechanism is unclear. This study found that the effect of ruminal acetate-to-propionate ratio was not affected by the concentration of the fermentation substrate, but was affected by the structure of the rumen microbiota. We believe that changes in the rumen microflora structure are the main mechanism for regulating the ruminal acetate-to-propionate ratio. This will help people to further understand the rumen physiology, thereby gradually improving feed conversion efficiency and reducing production costs. Abstract: In order to explore the mechanism by which diet regulates the acetate-to-propionate molar ratio (A: P ratio), we compared the effect on rumen fermentation parameters and the microbiome by altering the ratio of dietary concentrates to roughage ratio and calcium pyruvate infusion. The test animals were Laoshan dairy goats, and were fed continuously through an automatic feeder. The test groups were fed a base diet of low concentrates, and intraruminally infused with calcium pyruvate at two concentrations. The infusion concentrations were derived from the difference in the rate of carbohydrate degradation of the high and low concentrate diets, and they were artificially set such that the high concentration infusion group was infused with twice the concentration as the low concentration infusion group. The control groups were fed high concentrate (6:4) and low concentrate (3:7) diets, respectively. The following results were obtained by measuring rumen fermentation parameters and microbial composition: the rumen A: P ratio was significantly lower in the high-concentrate diet group than in the low concentrate diet group (P 0.05), while infusion of high concentration calcium pyruvate significantly increased the rumen A: P ratio (P effect on rumen microbial structure. The above results indicate that increasing the concentration of the fermentation substrate without affecting the composition of the microflora does not reduce the A: P ratio. Microbiological results showed that the A: P ratio was more closely related to the rumen microflora structure. Therefore, it is believed that rumen microflora structure is the main mechanism regulating A: P ratio in rumen fermentation.

Objectives of this study were to investigate the effects of supplementing rumen-protected methionine (RP-Met), threonine (RP-Thr), isoleucine (RP-Ile), and leucine (RP-Leu) individually or jointly to a low-protein diet, on the performance of lactating dairy cows, as well as to determine the effects of these amino acids (AA) on the mammalian target of rapamycin (mTOR) in vivo. Ten lactating Holstein cows were randomly allocated to a repeated 5 × 5 Latin square experiment with five 19-d periods. Treatments were high-protein diet (16% crude protein, positive control; HP), low-protein diet (12% crude protein, negative control; LP), LP plus RP-Met (LPM), LP plus RP-Met and RP-Thr (LPMT), and LP plus RP-Met, RP-Thr, RP-Ile, and RP-Leu (LPMTIL). The dry matter intakes (DMI) of the LP, LPM, and LPMT diets were lower than that of the HP diet, whereas the DMI of the LPMTIL diet was intermediate between the HP diet and the other LP diets. Supplementing RP-Met to the LP diet increased the yields of milk and milk protein, increased the content of milk urea N, and tended to increase milk N efficiency. Co-supplementation of RP-Thr with RP-Met resulted in no further milk production increase. Co-supplementation of all 4 rumen-protected amino acids (RP-AA) increased milk and lactose yields to the level of the HP diet and tended to increase milk protein yield compared with the LPMT diet. We found no significant differences in the contents and yields of milk components between the LPMTIL and HP diets except for a lower milk urea N content in the LPMTIL diet. Venous concentrations of the measured AA were similar across the LP and LP diets supplemented with RP-AA. Relative to levels of the HP diet, LP diets had higher venous concentrations of Met and Gly and tended to have higher Phe concentration and lower concentrations of Val and BCAA. The LPMTIL diet had higher venous concentrations of Arg, Lys, Met, Phe, and Glu, and a lower Val concentration. Phosphorylation status of the measured mTOR components in LPM and LPMT treatments were similar to those in the LP treatment but phosphorylation status of mTOR and eIF4E-binding protein 1 (4eBP1) in LPMTIL treatment were higher. The phosphorylation rates of eukaryotic elongation factor 2 (eEF2) in the 4 LP and LP plus RP-AA diets were higher than that of the HP diet. Overall, results of the present study supported the concept that under the relatively short time of this experiment, supplementing RP-AA, which are believed to stimulate the mTOR signal pathway, can lead to increased milk protein yield. This increase appears to be due to increased DMI, greater mTOR signaling, and greater eEF2 activity.

Non-alcoholic fatty liver disease (NAFLD) is rapidly being recognized as the leading cause of chronic liver disease worldwide. Men1, encoding protein of menin, is a key causative gene of multiple endocrine neoplasia type 1 syndrome including pancreatic tumor. It is known that insulin that secretes by endocrine tissue pancreatic islets plays a critical role in hepatic metabolism. Mouse model of hemizygous deletion of Men1 was shown to have severe hepatic metabolism disorders. However, the molecular function of menin on lipid deposition in hepatocytes needs to be further studied. Transcriptome sequencing does show that expression suppression of Men1 in mouse hepatocytes widely affect signaling pathways involved in hepatic metabolism, such as fatty acid metabolism, insulin response, glucose metabolism and inflammation. Further molecular studies indicates that menin overexpression inhibits expressions of the fat synthesis genes Srebp-1c, Fas, and Acc1, the fat differentiation genes Pparγ1 and Pparγ2, and the fat transport gene Cd36, thereby inhibiting the fat accumulation in hepatocytes. The biological process of menin regulating hepatic lipid metabolism was accomplished by interacting with the transcription factor FoxO1, which is also found to be critical for lipid metabolism. Moreover, menin responds to insulin in hepatocytes and mediates its regulatory effect on hepatic metabolism. Our findings suggest that menin is a crucial mediation factor in regulating the hepatic fat deposition, suggesting it could be a potential important therapeutic target for NAFLD.

The current study reports the identification of previously undiscovered single-nucleotide polymorphisms (SNPs) in the bovineAGPAT3gene and further investigates their associations with milk production traits. Our results demonstrate that the major allele C of the SNP g.12264 C > T is positively correlated with test-day milk yield, protein percentage and 305-day milk yield. Importantly,in silicoanalysis showed that the C/T transition at this locus gives rise to two new transcription factor binding sites (TFBS), E2F1 and Nkx3-2. Polymorphism g.18658 G > A was the only SNP associated with milk urea nitrogen (MUN) with the G allele related to an increase in milk urea nitrogen as well as fat percentage. The GG genotype of SNP g.28731 A > G was associated with the highest fat and protein percentage and lowest 305-day milk yield and somatic cell score (SCS). The association betweenAGPAT3locus and milk production traits could be utilized in marker-assisted selection for the genetic improvement of milk production traits and, probably in conjunction with other traits, for selection to improve fitness of dairy cattle.

Background Fatty liver disease prevalently occurs in commercial postpartum dairies, resulting in a worldwide high culling rate because of their subsequent limitations of production and reproduction performance. Results Fatty liver-specific proteome and acetylome analysis revealed that energy metabolism suppression closely associated with mitochondrial dysfunction and inflammation activation are shown to be remarkable biological processes underlying the development of fatty liver disease, thereby acetylation modification of proteins modulating these processes. Twenty pivotal genetic factors/genes that differentially expressing and being acetylation modified in liver were identified and proposed to regulate the pathogenesis of fatty liver dairies. These proteins were confirmed to be differentially expressing in individual liver tissues, eight of which being validated through immunohistochemistry assay. Conclusion This study provided a comprehensive proteome and acetylome profile of fatty liver of dairy cows, and revealed potential important biological processes and essential regulators in the pathogenesis of fatty liver disease. Expectantly, understanding the molecular mechanisms of the pathogenesis of fatty liver disease in dairies, as an animal model of non-alcoholic fatty liver disease (NAFLD) in human beings, which is a clinico-pathologically defined process associated with metabolic syndrome, could inspire and facilitate the development of efficacious therapeutic drugs on NAFLD.

Background: Drug-resistance and immunological escape of Staphylococcus aureus and its “superbug”, methicillin-resistant S. aureus (MRSA), have become one of main causes of bacterial infection in both human and animals. In dairy cattle, elimination of bovine mastitis induced by S. aureus is of importance because S. aureus-infected cows normally are culled passively. Methods: Here, we investigated the beneficial effects of bovine trafficking protein particle complex 9 (TRAPPC9) gene and folic acid supplementation in the control of mastitis induced by S. aureus or MRSA by a series of in vivo and in vitro experiments. Results: The data showed that the genetic mutations and DNA methylation of TRAPPC9 were highly linked with the mastitis resistance of dairy cows. Additionally, knockdown of bovine TRAPPC9 was significantly involved in the mRNA expression levels of interleukin’s genes (increased IL-1β and IL-6), and down-regulated the protein level of NF-κB-P65 in the mastitis cell model induced by MRSA. Meanwhile, dose-dependent folic acid addition can inhibit the invasion of MRSA into Mac-T cells and improve TRAPPC9 expression in dairy cows. Conclusions: Altogether, our data suggest that an appropriate dose of folic acid can significantly reduce the inflammation caused by MRSA partially through TRAPPC9 mediated NF-κB pathway. These findings provide new insights to control the drug-resistant pathogens and to restrict the overuse of antibiotics through combined effects of the intrinsic host gene and extrinsic nutrient.

A genome-wide association study (GWAS) was conducted on 23 serum biochemical traits in Chinese Holstein cattle. The experimental population consisted of 399 cattle, each genotyped by a commercial bovine 50K SNP chip, which had 49,663 SNPs. After data cleaning, 41,092 SNPs from 361 Holstein cattle were retained for GWAS. The phenotypes were measured values of serum measurements of these animals that were taken at 11 days after parturition. Two statistical models, a fixed-effect linear regression model (FLM) and a mixed-effect linear model (MLM), were used to estimate the association effects of SNPs. Genome-wide significant and suggestive thresholds were set up to be 1.22E-06 and 2.43E-06, respectively. In the Chinese Holstein population, FLM identified 81 genome-wide significant (0.05/41,092 = 1.22E-06) SNPs associated with 11 serum traits. Among these SNPs, five SNPs (BovineHD0100005950, ARS-BFGL-NGS-115158, BovineHD1500021175, BovineHD0800028900, and BTB-00442438) were also identified by the MLM to have genome-wide suggestive effects on CHE, DBIL, and LDL. Both statistical models pinpointed two SNPs that had significant effects on the Holstein population. The SNP BovineHD0800028900 (located near the gene LOC101903458 on chromosome 8) was identified to be significantly associated with serum high- and low-density lipoprotein (HDL and LDL), whereas BovineHD1500021175 (located in 73.4Mb on chromosome 15) was an SNP significantly associated with total bilirubin and direct bilirubin (TBIL and DBIL). Further analyses are needed to identify the causal mutations affecting serum traits and to investigate the correlation of effects for loci associated with fatty liver disease in dairy cattle.

Menin, the protein encoded by the MEN1 gene, is abundantly expressed in the epithelial cells of mammary glands. Here, we found MEN1/menin expression slowly decreased with advancing lactation but increased by the end of lactation. It happened that the number of bovine mammary epithelial cells decreases since lactation, suggesting a role of menin in the control of mammary epithelial cell growth. Indeed, reduction of menin expression through MEN1-specific siRNA transfection in the bovine mammary epithelial cells caused cell growth arrest in G1/S phase. Decreased mRNA and protein expression of Cyclin D1 was observed upon MEN1 knockdown. Furthermore, menin was confirmed to physically bind to the promoter region of Cyclin D1 through a ChIP assay, indicating that menin plays a regulatory role in mammary epithelial cell cycle progression. Moreover, lower expression of MEN1/menin induced increased epithelial cell apoptosis and caused extracellular matrix remodeling by down-regulating its associated genes, such as DSG2 and KRT5, suggesting that menin’s role may also be involved in the control of cell–cell adhesion in normal mammary glands. Taken together, our data revealed an unknown molecular function of menin in epithelial cell proliferation, which may be important in the regulation of lactation behavior of mammary glands. Electronic supplementary material The online version of this article (10.1007/s10911-017-9385-8) contains supplementary material, which is available to authorized users.

A genome-wide association study (GWAS) was conducted on 15 milk production traits in Chinese Holstein. The experimental population consisted of 445 cattle, each genotyped by the GGP (GeneSeek genomic profiling)-BovineLD V3 SNP chip, which had 26 151 public SNPs in its manifest file. After data cleaning, 20 326 SNPs were retained for the GWAS. The phenotypes were estimated breeding values of traits, provided by a public dairy herd improvement program center that had been collected once a month for 3 years. Two statistical models, a fixed-effect linear regression model and a mixed-effect linear model, were used to estimate the association effects of SNPs on each of the phenotypes. Genome-wide significant and suggestive thresholds were set at 2.46E-06 and 4.95E-05 respectively. The two statistical models concurrently identified two genome-wide significant (P

To identify the molecular effects of Tobacco bushy top virus (TBTV) evolution on the degeneration of tobacco bushy top disease, three TBTV isolates with mild virulence were compared with wild-type TBTV to assess the translation of p35, which relies on a BYDV-like translation element (BTE) in a cap-independent manner. The in vitro expression of p35 in the mild isolates was only 20% to 40% of the expression observed in wt TBTV. Based on translation data from chimeric TBTV RNA, low-level p35 expression in the three mild isolates was associated with two regions: the 5′ terminal 500 nt region (RI) and the 3′ internal region (RV), which included the BTE. For the RV region, low level p35 expression was mainly associated with structural alterations of the BTE instead of specific sequence mutations within the BTE based on SHAPE structural probing and mutation analysis. Additionally, structural alteration of the TBTV BTE resulted from mutations outside of the BTE, implying structural complexity of the local region surrounding the BTE. This study is the first report on the structural alteration of the 3′ cap-independent translation element among different isolates of a given RNA virus, which is associated with variations in viral virulence.

Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4.

Thirty healthy Holstein dairy cows in the dry period were randomly divided into three groups and fed diets with different net energy for lactation (group A: 1.2 Mcal/kg DM, group B: 1.3 Mcal/kg DM, and group C: 1.4 Mcal/kg DM) for 8 weeks prepartum. Thereafter, dairy cows were fed a diet of the same formulation (1.66 Mcal/kg DM) for 12 weeks postpartum. The effects of different dietary energy densities in the dry period on postpartum performance and metabolic parameters of dairy cows were observed. Milk yield was reduced by 14.5% in the low-energy diet group; however, there were no differences in milk composition between the three groups. Postpartum plasma β-hydroxybutyrate, non-esterified fatty acid, growth hormone, and glucagon levels were significantly decreased whereas leptin and neuropeptide levels were elevated in the low-energy diet group. Moreover, body fat mobilization was attenuated, and the decline in postpartum body condition was reduced in the low-energy diet group, thus effectively reducing the postpartum negative energy balance.

The present study investigated the effects of the supplemental dietary energy source on early lactating cows in a positive energy balance. Cows in the control group were fed a basal total mixed ration containing high-quality hay as forage, and the dietary concentrate to forage ratio was 45:65. The corn supplementation resulted in a significant decrease in the milk fat content (P < 0.05) and a low milk fat yield (P = 0.15), whereas the fat supplementation resulted in a decreasing trend of the milk protein and lactose content (P ≤ 0.1). Additionally, the corn supplementation significantly decreased the serum 5-hydroxytryptamine level (P < 0.05). The results support the proposal that the source of supplemental dietary energy has varying effects on feed intake, lactation performance, and the intermediate metabolism of early lactating cows in a positive energy balance. 5-Hydroxytryptamine secretion may be associated with the varying effects of the source of supplemental dietary energy.

MiR‐24‐3p, a broadly conserved, small, noncoding RNA, is abundantly expressed in mammary tissue. However, its regulatory role in this tissue remains poorly understood. It was predicted that miR‐24‐3p targets the 3′ untranslated region (3′‐UTR) of multiple endocrine neoplasia type 1 (MEN1), an important regulatory factor in mammary tissue. The objective of this study was to investigate the function of miR‐24‐3p in mammary cells. Using a luciferase assay in mammary epithelial cells (MAC‐T), miR‐24‐3p was confirmed to target the 3′‐UTR of MEN1. Furthermore, miR‐24‐3p negatively regulated the expression of the MEN1 gene and its encoded protein, menin. miR‐24‐3p enhanced proliferation of MAC‐T by promoting G1/S phase progression. MiR‐24‐3p also regulated the expression of key factors involved in phosphatidylinositol‐3‐kinase/protein kinase B/mammalian target of rapamycin and Janus kinase/signal transducer and activators of transcription signaling pathways, therefore controlling milk protein synthesis in epithelial cells. Thus, miR‐24‐3p appears to act on MAC‐T by targeting MEN1. The expression of miR‐24‐3p was controlled by MEN1/menin, indicating a negative feedback loop between miR‐24‐3p and MEN1/menin. The negatively inhibited expression pattern of miR‐24‐3p and MEN1 was active in mammary tissues at different lactation stages. The feedback mechanism is a new concept to further understand the lactation cycle of mammary glands and can possibly to be manipulated to improve milk yield and quality.

To determine whether insulin exerts an effect on milk fat yield through the direct regulation of milk fat synthesis in the mammary gland, the hyperinsulinemic-euglycemic clamp procedure was performed in lactating goats in the present study. The effects of the hyperinsulinemic-euglycemic clamp on milk yield, milk composition, milk fatty acid yield and the expression levels of mRNAs of milk fat synthesis-related genes were examined. The results revealed that the hyperinsulinemiceuglycemic clamp had no significant effect on the milk yield, the milk protein yield, the yield and content of lactose or the yield and content of solids-not-fat (SNF) (P > 0.05). In contrast, the milk fat percentage and milk fat yield were decreased by 35.3% and 33.6%, respectively (P C16) and 5 fatty acids synthesized de novo in the mammary gland ( 0.05), including acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), fatty acidbinding protein (FABP), lipoprotein lipase (LPL), stearoyl-CoA desaturase (SCD) and glycerol-3-phosphate acyltransferase (GPAT). However, the expression level of the SCD gene was significantly reduced during the post-procedure period (P

The mammary gland tissue exhibits a series of responses that are different from those of muscle and other peripheral tissues under amino acid deficiency. So, this present study was designed to investigate the effects of amino acid nutritional deficiency on the expression of protein metabolism-related genes in the mammary gland and muscle tissues of lactating mice. A total of 60 postpartum, lactating Kunming white mice were selected and randomly divided into 5 groups; each group contained 12 mice. Group A was the control group. The mice in group A were maintained on a normal diet after the initiation of lactation. Group B (starved) was given normal saline via intragastric administration. Group C (energy) was given glucose solution via intragastric administration. Groups D and E received a sodium caseinate solution via intragastric administration, which provided 0.5 g protein/d and 1.5 g protein/d, respectively. The results showed the following. 1) When the mice were exposed to nutritional stress caused by dietary amino acid deficiency, the β-casein mRNA expression level was increased in the mammary gland tissue. The increase in β-casein expression was the most significant in the energy-supplemented group, followed by the starved group (P P 14k and C2 (P P P < 0.05). 5) The phosphorylation level of p70S6K was elevated in the muscle tissues collected from the treatment groups. However, the magnitude of the increase was far smaller compared to that in the mammary gland tissues.

The MEN1 gene, which encodes the protein Menin, was investigated for its regulatory role in milk protein synthesis in mammary glands. Menin responds to nutrient and hormone levels via the PI3K/Akt/mTOR pathway. Bovine mammary epithelial cells and tissues were used as experimental models in this study. The results revealed that the milk protein synthesis capacity of mammary epithelial cells could be regulated by MEN1/Menin. The overexpression of Menin caused significant suppression of factors involved in the mTOR pathway, as well as milk protein κ-casein (CSNK). In contrast, a significant increase in these factors and CSNK was observed upon MEN1/Menin knockdown. The repression of MEN1/Menin on the mTOR pathway was also observed in mammary gland tissues. Additionally, MEN1/Menin was found to elicit a negative response on prolactin (PRL) and/or insulin (INS), which caused a similar downstream impact on mTOR pathway factors and milk proteins. Collectively, our data indicate that MEN1/Menin could play a regulatory role in milk protein synthesis through mTOR signaling in the mammary gland by mediating the effects of hormones and nutrient status. The discovery of Menin’s role in mammary glands suggests Menin could be potential new target for the improvement of milk performance and adjustment of lactation period of dairy cows.

The ovarian follicle supplies a unique dynamic system for gametes that ensures the propagation of the species. During folliculogenesis, the vast majority of the germ cells are lost or inactivated because of ovarian follicle atresia, resulting in diminished reproductive potency and potential infertility. Understanding the underlying molecular mechanism of folliculogenesis rules is essential. Primordial (P), preantral (M), and large antral (L) porcine follicles were used to reveal their genome-wide gene expression profiles. Results indicate that primordial follicles (P) process a diverse gene expression pattern compared to growing follicles (M and L). The 5,548 differentially expressed genes display a similar expression mode in M and L, with a correlation coefficient of 0.892. The number of regulated (both up and down) genes in M is more than that in L. Also, their regulation folds in M (2-364-fold) are much more acute than in L (2-75-fold). Differentially expressed gene groups with different regulation patterns in certain follicular stages are identified and presumed to be closely related following follicular developmental rules. Interestingly, functional annotation analysis revealed that these gene groups feature distinct biological processes or molecular functions. Moreover, representative candidate genes from these gene groups have had their RNA or protein expressions within follicles confirmed. Our study emphasized genome-scale gene expression characteristics, which provide novel entry points for understanding the folliculogenesis rules on the molecular level, such as follicular initiation, atresia, and dominance. Transcriptional regulatory circuitries in certain follicular stages are expected to be found among the identified differentially expressed gene groups.

The majority of the 3′ untranslated region (UTR) of Turnip crinkle virus (TCV) was previously identified as forming a highly interactive structure with a ribosome-binding tRNA-shaped structure (TSS) acting as a scaffold and undergoing a widespread conformational shift upon binding to RNA-dependent RNA polymerase (RdRp). Tertiary interactions in the region were explored by identifying two highly detrimental mutations within and adjacent to a hairpin H4 upstream of the TSS that reduce translation in vivo and cause identical structural changes in the loop of the 3′ terminal hairpin Pr. Second-site changes that compensate for defects in translation/accumulation and reverse the structural differences in the Pr loop were found in the Pr stem, as well as in a specific stem within the TSS and within the capsid protein (CP) coding region, suggesting that the second-site changes were correcting a conformational defect and not restoring specific base pairing. The RdRp-mediated conformational shift extended upstream through this CP open reading frame (ORF) region after bypassing much of an intervening, largely unstructured region, supporting a connection between 3′ elements and coding region elements. These data suggest that the Pr loop, TSS, and H4 are central elements in the regulation of translation and replication in TCV and allow for development of an RNA interactome that maps the higher-order structure of a postulated RNA domain within the 3′ region of a plus-strand RNA virus.

Plus-strand RNA viruses serve as templates for translation and then transcription by newly synthesized RdRp. A ribosome-binding tRNA-shaped structure (TSS) and upstream hairpin H4 in the 3′ UTR of Turnip crinkle virus (TCV) play key roles in translation and transcription. Second-site mutations generated to compensate for altering the critical asymmetric internal loop of H4 included a three- to two-base alteration in the terminal loop of a 3′ proximal hairpin (Pr) located downstream of the TSS. Unlike the non-deleterious three-base alteration, single mutations in Pr loop were detrimental for RdRp transcription while enhancing translation and RdRp binding. One deleterious mutation in the Pr loop altered the structures of both the TSS and H4. These complex interactions in the 3′ UTR support a compact structural arrangement likely permitting RdRp access to a number of residues within a 195-base region including the 3′ end that are necessary for efficient transcription initiation.

Primers used in this study. Primers were designed based on previously reported TSWV sequences. W: A/T, M: A/C, R: G/A, Y: C/T. (DOCX 15Â kb)

Summary of recombination events in different full-length TSWV L isolates using RDP4. NS: not significant. (DOCX 31Â kb)

Summary of recombination events in different full-length TSWV L isolates using RDP4. NS: not significant. (DOCX 33Â kb)

TSWV isolates used in this study. TSWV isolates from different countries use different abbreviations as followed, China (CL, CM and CS), South Korea (KL, KM and KS), Japan (JL, JM, JS), USA (UL, UM, US), Brazil (BL, BM, BS), Italy (IL, IM IS), Australia (AM), Spain (SM, SS), Germany (GS), Bulgaria (BuS). CS: Complementary sequence; N/A: Not analyzed. (DOCX 35Â kb)

Summary of recombination events in different full-length TSWV L isolates using RDP4. NS: not significant. (DOCX 24Â kb)

An effective isolation and identification method of primordial follicles would greatly benefit the animal production practice, transgenic animal production and endangered species conservation in the future. This study has not only advanced the isolation method but also developed an identification marker of primordial follicles. After enzymatic digestion, Percoll gradient centrifugation and mesh filtrations, the obtained follicular separations were then subjected to a cell sorter in order to collect primordial follicles. The study greatly improved the yield of primordial follicles (from about 1.85 x 10(5) to 7.79 x 10(5) per prepubertal ovary) by means of increasing cell layer number from 1 ml to 2.5 ml after Percoll gradient centrifugation. Based on traditional morphological criteria, the purity of recovered primordial follicles was averagely about 82.43+/-9.41% (n=5) because of their similar size and appearance with somatic cells. To further exactly appreciate the purity of sorted primordial follicles, a germ cell-specific protein, MSY2, was used to recognize the oocytes of primordial follicles. The results of repeat experiments showed that about 98.31+/-0.73% (n=4) of the primordial follicles cluster was MSY2-positive, which indicated the identification method of primordial follicles was more effective and high-yielded than the previous methods because of its higher purity.

Turnip crinkle virus (TCV) contains a structured 3′ region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3′ untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE ( s elective 2′- h ydroxyl a cylation analyzed by p rimer e xtension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3′UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. IMPORTANCE Genomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3′ UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3′ UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.

Background Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. Methods RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. Results Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5′ regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. Conclusions All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative-sense RNA viruses. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0457-3) contains supplementary material, which is available to authorized users.

Background During the past decade, tobacco bushy top disease, which is mainly caused by a combination of Tobacco bushy top virus (TBTV) and Tobacco vein-distorting virus (TVDV), underwent a sudden appearance, extreme virulence and degeneration of the epidemic in the Yunnan province of China. In addition to integrative control of its aphid vector, it is of interest to examine diversity and evolution among different TBTV isolates. Methods 5’ and 3’ RACE, combined with one step full-length RT-PCR, were used to clone the full-length genome of three new isolates of TBTV that exhibited mild pathogenicity in Chinese fields. Nucleotide and amino acid sequences for the TBTV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. Results The genomes of three isolates, termed TBTV-JC, TBTV-MD-I and TBTV-MD-II, were 4152 nt in length and included one distinctive difference from previously reported TBTV isolates: the first nucleotide of the genome was a guanylate instead of an adenylate. Diversity and phylogenetic analyses among these three new TBTV isolates and five other available isolates suggest that ORFs and 3’UTRs of TBTV may have evolved separately. Moreover, the RdRp-coding region was the most variable. Recombination analysis detected a total of 29 recombination events in the 8 TBTV isolates, in which 24 events are highly likely and 5 events have low-level likelihood based on their correlation with the phylogenetic trees. The three new TBTV isolates have individual recombination patterns with subtle divergences in parents and locations. Conclusions The genome sizes of TBTV isolates were constant while different ORF-coding regions and 3’UTRs may have evolved separately. The RdRp-coding region was the most variable. Frequent recombination occurred among TBTV isolates. Three new TBTV isolates have individual recombination patterns and may have different progenitors. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0340-2) contains supplementary material, which is available to authorized users.

Melanocortin receptor 1 (MC1R) gene, one of the important candidate genes for coat color trait, was used to analyze the single nucleotide polymorphism (SNP) in Chinese native pig breeds by PCR-single strand conformation polymorphism (PCR-SSCP). The study had also taken 3 imported pig breeds as control. The results showed that the three mutations G284A, T309C and T364C found in Chinese native pigs were consistent to the mutation found in the European Large Black individuals. However, 68CC or C492T and G728A were only found in the imported individuals, which were obviously different from the Chinese native pigs. Accordingly, we presumed that the coat colors of Chinese native pigs belonged to dominant black color system, which was completely distinct to that of imported pig breeds. Thus it was implied that MC1R gene was not the principal factor affecting the coat color differences of Chinese native pig breeds, but could be used to trace the molecular evolution of pig breeds.

To determine the effects of the initial timing of forage supply on growth, ruminal fermentation parameters and rumen development in preweaning calves, 18 7-day-old Holstein calves of 42 ± 3 kg were randomly divided into three treatment groups. The dietary treatments were (1) milk and commercial starter diet (MS) control, (2) milk and starter diet supplemented with oat hay at 2 weeks (MSO2), and (3) milk and starter diet supplemented with oat hay at 6 weeks (MSO6). Starter feed and oat hay were provided ad libitum, and 2 L of milk was provided twice daily. Samples were collected at 64 days of age. Supplementing with hay increased the dry-matter intake; P < 0.05). The height, body length, heart girth and cannon bone circumference were similar among the three treatments (P > 0.05). The concentrations of serum glucose (P = 0.07) and β-hydroxybutyric acid; P < 0.05) were lower in the MSO6 group than in the MS and MSO2 groups. Rumination time was longer, and time spent on non-nutritive oral behaviour (such as e.g. licking surfaces, tongue rolling, wood-shaving consumption) was lower for hay-supplemented calves than for the control (P < 0.05). Although ruminal pH of hay-supplemented calves was significantly higher than that of the control, total rumen fluid volatile fatty acid concentrations were not significantly different among treatments. Calves in the MSO2 group had a smaller ratio of empty weight to slaughter weight (P < 0.05) and a larger total digestive tract weight (P < 0.05); the empty gastrointestinal tract weights were similar among the three treatments, suggesting that MSO2 calf weight gain may have resulted from intestinal chyme accumulation. Compared with the control, hay-supplemented calves had reduced rumen papilla width and epithelium thickness (P < 0.05), and no discernable plaque formation. Hay supplementation in the diet of preweaning calves improved the overall dry-matter intake, improved rumination, reduced non-nutritive oral behaviours, improved rumen pH, and ensured healthy rumen development; furthermore, productivity and rumen development were better in calves supplemented with hay from the second week.

An animal feeding trial was conducted on 18 seven‐day‐old Holstein dairy bull calves weighing 42 ± 3 kg each. Calves were randomly assigned into three groups (n = 6 each). The dietary treatments were as follows: (1) milk and starter for the control group (MS), (2) supplementation of oat hay from week 2 on the basis of milk and starter (MSO2), and (3) supplementation of oat hay from week 6 on the basis of milk and starter (MSO6). All animals were fed starter and oat hay ad libitum. The major phyla in the different groups of rumen fluid included Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Euryarchaeota. The major genera were identified, and major genera proportions in the three groups were as follows: Methanobrevibacter (Euryarchaeota), 2.1%, 1.7%, and 2.1%; Olsenella (Actinobacteria), 23.9%, 17.7%, and 12.8%; Prevotella (Bacteroidetes), 10.5%, 16.5%, and 19.2%; Dialister (Firmicutes), 3.3%, 4.1%, and 2.8%; Succiniclasticum (Firmicutes), 3.8%, 4.7%, and 9.2%; and Sharpea (Firmicutes), 0.4%, 2.5%, and 0.2%, respectively. There were no significant differences in the various phyla among the three groups (p > .05). The results showed that calves hay supplementation time did not affect the diversity of the rumen microbiota in the suckling calves. However, the hay supplementation altered the proportion of the various microbial populations, supplementation of oat hay from week 2 on the basis of milk and starter could improve calves rumen pH.

The multiple endocrine neoplasia type 1 (MEN1) syndrome is caused by germline mutations in the MEN1 gene encoding menin, with tissue-specific tumors of the parathyroids, anterior pituitary, and enteropancreatic endocrine tissues. Also, 30–40% of sporadic pancreatic endocrine tumors show somatic MEN1 gene inactivation. Although menin is expressed in all cell types of the pancreas, mouse models with loss of menin in either pancreatic α-cells, or β-cells, or total pancreas develop β-cell-specific endocrine tumors (insulinomas). Loss of widely expressed tumor suppressor genes may produce tissue-specific tumors by reactivating one or more embryonic-specific differentiation factors. Therefore, we determined the effect of menin overexpression or knockdown on the expression of β-cell differentiation factors in a mouse β-cell line (MIN6). We show that the β-cell differentiation factor Hlxb9 is posttranscriptionally upregulated upon menin knockdown, and it interacts with menin. Hlxb9 reduces cell proliferation and causes apoptosis in the presence of menin, and it regulates genes that modulate insulin level. Thus, upon menin loss or from other causes, dysregulation of Hlxb9 predicts a possible combined mechanism for β-cell proliferation and insulin production in insulinomas. These observations help to understand how a ubiquitously expressed protein such as menin might control tissue-specific tumorigenesis. Also, our findings identify Hlxb9 as an important factor for β-cell proliferation and insulin regulation.