Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis.

BACKGROUND: Milk is a complex liquid that provides nutrition to newborns. Recent reports demonstrated that milk is enriched in maternal-derived exosomes that are involved in fetal physiological and pathological conditions by transmission of exosomal mRNAs, miRNAs and proteins. Until now, there is no such research relevant to exosomal mRNAs and proteins in porcine milk, therefore, we have attempted to investigate porcine milk exosomal mRNAs and proteins using RNA-sequencing and proteomic analysis. RESULTS: A total of 16,304 (13,895 known and 2,409 novel mRNAs) mRNAs and 639 (571 known, 66 candidate and 2 putative proteins) proteins were identified. GO and KEGG annotation indicated that most proteins were located in the cytoplasm and participated in many immunity and disease-related pathways, and some mRNAs were closely related to metabolisms, degradation and signaling pathways. Interestingly, 19 categories of proteins were tissue-specific and detected in placenta, liver, milk, plasma and mammary. COG analysis divided the identified mRNAs and proteins into 6 and 23 categories, respectively, 18 mRNAs and 10 proteins appeared to be involved in cell cycle control, cell division and chromosome partitioning. Additionally, 14 selected mRNAs were identified by qPCR, meanwhile, 10 proteins related to immunity and cell proliferation were detected by Western blot. CONCLUSIONS: These results provide the first insight into porcine milk exosomal mRNA and proteins, and will facilitate further research into the physiological significance of milk exosomes for infants.

Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

Distribution and linkage disequilibrium analysis of polymorphisms of MC4R, LEP, H-FABP genes in the different populations of pigs, associated with economic traits in DIV2 line.

PCR-RFLP was used to analyze the polymorphisms of MC4R, LEP, H-FABP genes in a swine breed composite (DIV2) and 4 swine breeds (Yorkshire, Landrace, Meishan, Bamei). The association study of these polymorphisms with several economic traits was carried out on a DIV2 population. The results obtained showed that MC4R/TaqI genotype had an effect for average backfat thickness (P < 0.05) and lean meat percentage (P < 0.05). At locus LEP/HinfI animals of AA genotype had lower test daily gain than that of BB (P < 0.01) or AB genotype (P < 0.05). At the H-FABP/HaeIII locus lean meat percentage of the individuals with genotype DD were higher than that with genotype dd (P < 0.05). Linkage disequilibrium analysis among MC4R, LEP and H-FABP revealed that these genes were independent. This represented two or more genes that could be combined together within one genotype in order to facilitate breeding for objective traits. In addition, a method allowing simultaneous detection of fragments of MC4R and LEP gene was developed.

MiR-125b-2 knockout increases high-fat diet-induced fat accumulation and insulin resistance.

Obese individuals are more susceptible to comorbidities than individuals of healthy weight, including cardiovascular disease and metabolic disorders. MicroRNAs are a class of small and noncoding RNAs that are implicated in the regulation of chronic human diseases. We previously reported that miR-125b plays a critical role in adipogenesis in vitro. However, the involvement of miR-125b-2 in fat metabolism in vivo remains unknown. In the present study, miR-125b-2 knockout mice were generated using CRISPR/CAS9 technology, resulting in mice with a 7 bp deletion in the seed sequence of miR-125b-2. MiR-125b-2 knockout increased the weight of liver tissue, epididymal white fat and inguinal white fat. MiR-125b-2 knockout also increased adipocyte volume in HFD-induced obese mice, while there were no significant differences in body weight and feed intake versus mice fed a normal diet. Additionally, qRT-PCR and western blot analysis revealed that the expression of the miR-125b-2 target gene SCD-1 and fat synthesis-associated genes, such as PPARγ and C/EBPα, were significantly up-regulated in miR-125b-2KO mice (P < 0.05). Moreover, miR-125b-2KO altered HFD-induced changes in glucose tolerance and insulin resistance. In conclusion, we show that miR-125b-2 is a novel potential target for regulating fat accumulation, and also a candidate target to develop novel treatment strategies for obesity and diabetes.

Genotype identification and phylogenetic analysis of Enterocytozoon bieneusi in farmed black goats (Capra hircus) from China's Hainan Province.

Enterocytozoon bieneusi is an important pathogen commonly found in humans and animals. Farmed animals with close contact to humans are important hosts of E. bieneusi. The role of goats in the transmission of E. bieneusi, however, remains unclear. In this study, 341 fresh fecal samples of black goats were collected from five locations in Hainan Province, China. Enterocytozoon bieneusi was identified and genotyped by sequences of the internal transcribed spacer (ITS) region. Phylogenetic analysis was performed by constructing a neighbor-joining tree of the ITS gene sequences. The average prevalence of E. bieneusi in black goats was 24.0% (82/341) with rates ranging from 6.3% (4/63) to 37.2% (32/86) across the locations (χ2 = 17.252, p < 0.01). Eight genotypes of E. bieneusi were identified, including six known genotypes: CHG5 (n = 47); CHG3 (n = 23); CHG2 (n = 4); CM21 (n = 3); D (n = 2); and AHG1 (n = 1), and two novel genotypes termed HNG-I (n = 1) and HNG-II (n = 1). In the phylogenetic tree, genotype D was clustered into Group 1 and the other identified genotypes were included in Group 2. This represents the first report identifying E. bieneusi in black goats from Hainan Province, with a high prevalence and wide occurrence demonstrated. The two new genotypes identified provide additional insights into the genotypic variations in E. bieneusi. Due to the small percentage of zoonotic genotypes in these animals, there is minimal risk of zoonotic transmission of E. bieneusi.

TITLE: Identification du génotype et analyse phylogénétique d'Enterocytozoon bieneusi chez des chèvres noires (Capra hircus) de la province de Hainan, en Chine. ABSTRACT: Enterocytozoon bieneusi est un agent pathogène important que l'on trouve couramment chez l'homme et les animaux. Les animaux d'élevage, en contact étroit avec l'homme, sont des hôtes importants d'E. bieneusi. Le rôle des chèvres dans la transmission d'E. bieneusi reste toutefois incertain. Dans cette étude, 341 échantillons de fèces fraîches de chèvres noires ont été prélevés dans cinq sites de la province de Hainan, en Chine. Enterocytozoon bieneusi a été identifié et génotypé par des séquences de la région de l'espaceur interne transcrit (ITS). L'analyse phylogénétique a été réalisée en construisant un arbre de jonction voisine des séquences du gène ITS. La prévalence moyenne d'E. bieneusi chez les chèvres noires était de 24,0 % (82/341), avec des taux allant de 6,3 % (4/63) à 37,2 % (32/86) dans tous les sites (χ2 = 17,252, p < 0,01). Huit génotypes d'E. bieneusi ont été identifiés, dont six génotypes connus: CHG5 (n = 47) ; CHG3 (n = 23) ; CHG2 (n = 4) ; CM21 (n = 3) ; D (n = 2) ; AHG1 (n = 1) et deux nouveaux génotypes appelés HNG-I (n = 1) et HNG-II (n = 1). Dans l'arbre phylogénétique, le génotype D appartenait au groupe 1 et les autres génotypes identifiés étaient inclus dans le groupe 2. Il s'agit du premier rapport identifiant E. bieneusi chez des chèvres noires de la province de Hainan, avec une prévalence élevée et une occurrence étendue. Les deux nouveaux génotypes identifiés fournissent des informations supplémentaires sur les variations génotypiques chez E. bieneusi. En raison du faible pourcentage de génotypes zoonotiques chez ces animaux, le risque de transmission zoonotique d'E. bieneusi est minime.

In low protein diets, microRNA-19b regulates urea synthesis by targeting SIRT5.

Ammonia detoxification, which takes place via the hepatic urea cycle, is essential for nitrogen homeostasis and physiological well-being. It has been reported that a reduction in dietary protein reduces urea nitrogen. MicroRNAs (miRNAs) are major regulatory non-coding RNAs that have significant effects on several metabolic pathways; however, little is known on whether miRNAs regulate hepatic urea synthesis. The objective of this study was to assess the miRNA expression profile in a low protein diet and identify miRNAs involved in the regulation of the hepatic urea cycle using a porcine model. Weaned 28-days old piglets were fed a corn-soybean normal protein diet (NP) or a corn-soybean low protein diet (LP) for 30 d. Hepatic and blood samples were collected, and the miRNA expression profile was assessed by sequencing and qRT-PCR. Furthermore, we evaluated the possible role of miR-19b in urea synthesis regulation. There were 25 differentially expressed miRNAs between the NP and LP groups. Six of these miRNAs were predicted to be involved in urea cycle metabolism. MiR-19b negatively regulated urea synthesis by targeting SIRT5, which is a positive regulator of CPS1, the rate limiting enzyme in the urea cycle. Our study presented a novel explanation of ureagenesis regulation by miRNAs.

Porcine milk-derived exosomes promote proliferation of intestinal epithelial cells.

Milk-derived exosomes were identified as a novel mechanism of mother-to-child transmission of regulatory molecules, but their functions in intestinal tissues of neonates are not well-studied. Here, we characterized potential roles of porcine milk-derived exosomes in the intestinal tract. In vitro, treatment with milk-derived exosomes (27 ± 3 ng and 55 ± 5 ng total RNA) significantly promoted IPEC-J2 cell proliferation by MTT, CCK8, EdU fluorescence and EdU flow cytometry assays. The qRT-PCR and Western blot analyses indicated milk-derived exosomes (0.27 ± 0.03 µg total RNA) significantly promoted expression of CDX2, IGF-1R and PCNA, and inhibited p53 gene expression involved in intestinal proliferation. Additionally, six detected miRNAs were significantly increased in IPEC-J2 cell, while FAS and SERPINE were significantly down-regulated relative to that in control. In vivo, treated groups (0.125 µg and 0.25 µg total RNA) significantly raised mice' villus height, crypt depth and ratio of villus length to crypt depth of intestinal tissues, significantly increased CDX2, PCNA and IGF-1R' expression and significantly inhibited p53' expression. Our study demonstrated that milk-derived exosomes can facilitate intestinal cell proliferation and intestinal tract development, thus giving a new insight for milk nutrition and newborn development and health.