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.

Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China.

To understand the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in South China, we collected 231 clinical samples from pigs with suspected PRRSV infection in Guangdong between 2007 and 2009. We found that 74 of 231 samples were positive by RT-PCR. The PCR products of the ORF5 gene of 35 isolates from different farms were sequenced and their DNA sequences were compared to 23 other PRRSV isolates in the GenBank. We found that the nucleotide similarity among all South China isolates ranged from 87.6% to 100%, and all belonged to the North American genotype. Most of them were classified into subgenotype I, but the rest mapped to subgenotypes III, V or VI. Those in subgenotypes I and III were found to be highly variable in the primary neutralising epitope (PNE) with a specific amino acid mutation (F39/L39→I39), and a few isolates in subgenotypes I and III isolates also had a mutation at L41 (L41→S41). PRRSV isolates in subgenotypes III, V and VI had less potential glycosylation sites than those in subgenotype I. Our data contribute to the understanding of molecular variation of PRRSV in South China.

Effect of compounds on the purification and antibody preparation of the extracellular domain fragment of the receptor CD163.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) has been acknowledged as one of the most important agents affecting swine. The scavenger receptor CD163 is one of the important entry mediators for PRRSV. RESULTS: The tD4 and tD5 CD163 genes were amplified, and the PCR products were cloned into pET-28a(+) (designated pET-28a-tD4 and pET-28a-tD5, respectively). The plasmids pET-28a-tD4 and pET-28a-tD5 were then transformed into the E. coli BL21 (DE3) strain and expressed by adding 1 mmol/L of isopropyl-beta-D-thiogalactopyranoside. The proteins were highly expressed in the supernatant from the tD4- and tD5-producing cells that were incubated with a binding buffer containing the following compounds: ß-mercaptoethanol, urea, Tween 20, glycerol, and SDS, while they were rarely expressed in the supernatant from the tD4- and tD5-producing cells that were incubated with binding buffer without the compounds. The tD4 and tD5 proteins were purified, and BALB/c mice were immunized with the purified proteins. Western blotting analysis showed that the tD4 and tD5 proteins were capable of reacting with tD5 antibodies; the titer of both the tD4 and tD5 antiserums was 1:160 against the tD5 protein, as shown by ELISA. CONCLUSIONS: These studies provide a new way for the purification of proteins expressed in inclusion bodies and the preparation of the corresponding antibodies.

Genotyping and zoonotic potential of Enterocytozoon bieneusi in cattle farmed in Hainan Province, the southernmost region of China.

Enterocytozoon bieneusi is an intestinal pathogen that infects a wide range of species, including humans. Cattle constitute an important host for E. bieneusi; however, there is a scarcity of information on the prevalence and genotyping of E. bieneusi in cattle in the Hainan Province of China. In this study, PCR analysis of 314 fecal samples from cattle in six cities of Hainan was performed for genotype identification. The average prevalence of E. bieneusi in these animals was 9.9% (31/314), and ranged from 0.0% (0/12) to 20.5% (8/39). Five known genotypes - EbpC (n = 14), BEB4 (n = 12), J (n = 2), I (n = 1), and CHG5 (n = 1) - and a novel genotype: HNC-I (n = 1) - were identified. Genotypes EbpC and HNC-I were placed in zoonotic Group 1, and the remaining four genotypes (BEB4, J, I, and CHG5) were placed in Group 2. Since 93.5% of the genotypes found in the cattle (29/31) (EbpC, BEB4, J, and I) have previously been found in humans, these genotypes are probably involved in the transmission of microsporidiosis to humans.

TITLE: Génotypage et potentiel zoonotique d'Enterocytozoon bieneusi chez les bovins élevés dans la province de Hainan, la région la plus au sud de la Chine. ABSTRACT: Enterocytozoon bieneusi est un pathogène intestinal qui infecte un large éventail d'espèces, y compris les humains. Le bétail constitue un hôte important pour E. bieneusi, mais les informations sur la prévalence et le génotypage d'E. bieneusi chez les bovins de la province de Hainan en Chine sont rares. Dans cette étude, une analyse PCR de 314 échantillons fécaux provenant de bovins dans six villes de Hainan a été réalisée pour l'identification du génotype. La prévalence moyenne d'E. bieneusi chez ces animaux était de 9,9 % (31/314), et variait de 0,0 % (0/12) à 20,5 % (8/39). Cinq génotypes connus, EbpC (n = 14), BEB4 (n = 12), J (n = 2), I (n = 1) et CHG5 (n = 1), et un nouveau génotype, HNC-I (n = 1), ont été identifiés. Les génotypes EbpC et HNC-I sont placés dans le groupe zoonotique 1, et les quatre génotypes restants (BEB4, J, I et CHG5) sont placés dans le groupe 2. Puisque 93,5 % (29/31) (EbpC, BEB4, J et I) des génotypes trouvés chez les bovins ont déjà été trouvés chez l'homme, ces génotypes sont probablement impliqués dans la transmission de la microsporidiose à l'homme.

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.