The super-enhancer repertoire in porcine liver.

The transcriptional initiation of genes is inextricably bound with the functions of cis-regulatory sequences. The pig is one of the most important livestock species and an ideal animal model for biomedical studies. At the same time, the liver is a critical organ with diverse and complex metabolic functions. Here, we performed Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with high-throughput sequencing to profile the chromatin landscape of histone H3 lysine 27 acetylation (H3K27ac), histone H3 lysine 4 monomethylation (H3K4me1), and CCAAT enhancer-binding protein ß (C-EBPß) in the 70-d-old porcine liver, compared the different profiles among the three markers and their associated stitched-enhancers by stitching and sorting the peaks within 12.5 kb (Pott and Lieb, 2015) and generated the porcine liver-specific super-enhancers (SEs) by the combination of three markers. Compared to typical enhancers (TEs) and other stitched-enhancers, liver-specific SEs showed a higher density of cis-motifs and SNPs, which may recruit more tissue-specific vital TFs. The expression profiles in fetal and 70-d-old pigs proved that a large proportion of SE-associated genes were up-regulated and were more related to hepatic metabolisms and detoxification pathways. Our results illustrated the difference and connection among promoter and enhancer markers, identified the features of liver SEs and their associated genes, and provided novel insight into cis-element identification, function, and liver transcriptional regulation.

The cis-regulatory elements including promoters, enhancers, and newly identified super-enhancers (SEs), which were reported to function both promoter and enhancer capabilities, play critical roles in selective gene expression during development and disease. To reveal and compare the characteristics of these cis-elements in liver, we first performed a genome-wide profile of H3K27ac, H3K4me1, and C-EBPß, then constructed their associated stitched-enhancers respectively. Porcine liver-specific SEs were generated by overlapping the three stitched-enhancers. The genomic and genic location, TF binding sites and SNP distribution patterns were compared among these cis-elements. We found that stitched-enhancers gather in regions with higher gene densities and locate closer to the transcription starting sites. Additionally, SEs showed higher density of TF binding sites and SNPs. To access the transcriptional consequences of liver SEs, we first analyzed the genes locationally associated with SEs. The KEGG results suggested that these genes are significantly involved in metabolisms, detoxification, and autophagy pathways. We also detected the liver gene expression profiles using RNA-seq and noticed that SE-associated genes are more likely to be up-regulated. Our results provided novel information on the identification, function, and transcriptional regulation of cis-elements in the liver.

Dietary mulberry-leaf flavonoids supplementation improves liver lipid metabolism and ovarian function of aged breeder hens.

Mulberry-leaf flavonoids (MF), extracted from mulberry leaves, exert antioxidant and hypolipidemic effects. The purpose of this experimental study was to investigate the effects of dietary MF on the ovarian function and liver lipid metabolism of aged breeder hens. We used 270 (60-weeks-old) Qiling breeder hens randomly assigned in 3 treatments with supplemental dietary MF doses (0, 30, 60 mg/kg). The results showed that dietary MF significantly improved the egg-laying rate, followed by the reduced feed conversion rate (FCR) (p < 0.05). However, there is no obvious difference in hatchability and fertilised eggs hatchability among the three groups (p > 0.05). The level of T-CHO, LDL-C and AKP in serum was reduced, and the HDL-C concentrations were increased by dietary MF (p < 0.05). MF treatment also improved the antioxidant capacity and reduced the apoptotic index of the ovary (p < 0.05). Additionally, dietary MF significantly increased the serum estradiol (E2) levels (p < 0.05) and the transcription level of CYP19A1 and LHR in the ovary (p < 0.05). Dietary MF enhanced fatty acid ß-oxidation in the liver via up-regulating the mRNA expressions of PPARα and CPT-I (p < 0.05). Moreover, the HMF group significantly decreased mRNA expressions of SREBP-1c (p < 0.05) and increased mRNA expressions of ERα, VTG-Ⅱ and ApoB in the liver (p < 0.05). In conclusion, dietary MF could improve the reproduction performance of aged breeder hens through improving ovary function and hepatic lipid metabolism.

Dietary mulberry-leaf flavonoids improve the eggshell quality of aged breeder hens.

Eggshell quality is subject to a significant decline in the late laying period, which results in huge economic losses. The purpose of this study was to investigate the effects of dietary mulberry-leaf flavonoids (MF) on the eggshell quality of aged breeder hens. A total of 270 (60-week-old) Qiling breeder hens were randomly assigned to 3 treatments with supplemental dietary MF doses (0, 30, and 60 mg/kg). The results showed that dietary MF improved the eggshell thickness and strength, following the reduced broken egg ratio (P < 0.05). Histological analysis showed that dietary MF increased glandular density and luminal epithelium height in the shell gland (P < 0.05). MF treatment reduced the apoptotic index of the shell gland, following by improved antioxidant capacity (P < 0.05). The protein expression of Caspase 3 was down-regulated, and Nrf2 was up-regulated by dietary MF (P < 0.05). Furthermore, calcium (Ca) content in the serum and shell gland, as well as the activity of Ca2+-ATPase in the shell gland were increased by dietary MF (P < 0.05). Ca transport-related genes (ESRα, ESRß, KCNA1, OPN, CABP-28K and CDH6) in the shell gland were upregulated by dietary MF treatment (P < 0.05). In conclusion, dietary MF could ameliorate the eggshell quality of aged hens by improving antioxidative capability and Ca deposition in the shell gland of uterus.

Dietary hawthorn-leaves flavonoids improves ovarian function and liver lipid metabolism in aged breeder hens.

Hawthorn-leaves flavonoids (HF), extracted from hawthorn leaves, were reported to exert antioxidant, anti-inflammatory and hypolipidemic properties. The aim of our study was to investigate the effects of dietary HF on the reproduction performance and liver lipid metabolism of aged breeder hens. A total of 270 aged Qiling breeder hens (60-wk-old) were randomly divided into 3 treatments: 1) basic corn-soybean diet (CON); 2) basic corn-soybean diet supplemented with 30 mg/kg HF (LHF); 3) basic corn-soybean diet supplemented with 60 mg/kg HF (HHF). The results showed that supplemented HF significantly improved the egg-laying rate and hatching rate of aged breeder hens (P < 0.05). HF treatment reduced the serum TG, T-CHO and L-LDL levels (P < 0.05), and upregulated the mRNA expressions of ESR1, ESR2, VTGⅡ, ApoB, and ApoVI in the liver (P < 0.05). Serum estrogen levels in HF treated groups were elevated compared with the CON group (P < 0.05). In the HHF group, the number of the primordial follicles was higher in comparison with the CON group (P < 0.05). Furthermore, dietary supplementation with HF improved the activity of antioxidant enzymes (T-AOC, GSH-Pχ) (P < 0.05), following with the reversed ovarian apoptosis and morphological damage. In addition, 60 mg/kg dietary HF upregulated the protein expression of PCNA and Nrf2 in the ovary (P < 0.05). In summary, dietary supplementation with HF could improve the reproduction performance through regulating liver lipid metabolism and improving ovarian function in aged breeder hens.

Drinking Water with Saccharin Sodium Alters the Microbiota-Gut-Hypothalamus Axis in Guinea Pig.

The effects of saccharin, as a type of sweetener additive, on the metabolism and development of mammals are still controversial. Our previous research revealed that saccharin sodium (SS) promoted the feed intake and growth of guinea pigs. In this experiment, we used the guinea pig model to study the physiological effect of SS in the microbiota-gut-hypothalamus axis. Adding 1.5 mM SS to drinking water increased the serum level of glucose, followed by the improvement in the morphology and barrier function of the ileal villus, such as SS supplementation which increased the villus height and villus height/crypt depth ratio. Saccharin sodium (SS) treatment activated the sweet receptor signaling in the ileum and altered GHRP hormone secretion. In the hypothalamus of SS and control (CN) group, RNA-seq identified 1370 differently expressed genes (796 upregulated, 574 downregulated), enriching into the taste signaling transduction, and neuroactive ligand-receptor interaction. LEfSe analysis suggested that Lactobacillaceae-Lactobacillus was the microbe with significantly increased abundance of ileum microorganisms in the SS-treated group, while Brevinema-Andersonii and Erysipelotrichaceae-Ilebacterium were the microbes with significantly increased abundance of the control. Furthermore, SS treatment significantly enhanced the functions of chemoheterotrophy and fermentation of ileal microflora compared to the CN group. Accordingly, SS treatment increased levels of lactic acid and short-chain fatty acids (acetic acid, propionic acid and N-valeric acid) in the ileal digesta. In summary, drinking water with 1.5 mM SS activated sweet receptor signaling in the gut and altered GHRP hormone secretion, followed by the taste signaling transduction in the hypothalamus.

Dietary alpha-lipoic acid supplementation improves spermatogenesis and semen quality via antioxidant and anti-apoptotic effects in aged breeder roosters.

The purpose of the present study was to investigate the effects of dietary alpha-lipoic acid (ALA) supplementation on the reproductive performance of aged breeder roosters. Sixteen 50-wk-old ROSS 308 breeder roosters were randomly allocated to two groups: roosters received a basal diet (CON), or a basal diet supplemented with 300 mg/kg of ALA (ALA). The results indicated that dietary ALA supplementation significantly increased sperm concentration, motility, viability, and membrane functional integrity. ALA also dramatically increased seminiferous tubule epithelial height (SEH) and testis scores. The ALA group had a higher serum concentration of testosterone than the CON group. ALA supplementation remarkably increased total antioxidant capacity (T-AOC), the enzyme activities of glutathione peroxidase (GPx), and catalase (CAT) in the testes; following a decrease in malondialdehyde (MDA) levels. In addition, we noted significant upregulation of Nrf2 mRNA and protein expression of and mRNA expression of its Downstream Genes (GPx1, NQO1, and GCLC), as well as significant downregulation of Keap1 mRNA expression in testicular tissue of aged roosters with ALA supplementation. The protein expression of Caspase 3 was downregulated and the protein expression of proliferating cell nuclear antigen (PCNA) was upregulated by ALA supplementation. The mRNA expression of spermatogenesis-related genes (ER1, AKT1, and Cav1) were markedly augmented in the ALA group compared with the CON group. In conclusion, dietary ALA supplementation enhanced the testicular antioxidant capacity through the Nrf2-signaling pathway, exerted anti-apoptotic effects, and improved the reproductive performance of aged roosters.

Alpha-lipoic acid improves the reproduction performance of breeder hens during the late egg-laying period.

Alpha-lipoic acid (ALA), a multifunctional antioxidant, can promote fatty acid mobilization, energy expenditure and scavenge free radicals. The effects of dietary ALA on the reproductive performance of breeder hens were investigated in the current study. In the 5-week experiment, 180 54-week-old Qiling breeder hens were randomly divided into three treatments with five replicates and supplemented with three levels of ALA (0, 300 and 600 mg/kg) in the basic corn-soya bean meal diets. 600 mg/kg ALA treatment group (HLA) significantly improved the eggshell thickness and strength (p < .05). ALA-treated groups improved egg-laying rate compared with the CON group, but with no statistically significant difference (p > .05). The levels of HDL-C, ALB and estradiol (E2) of the serum in the HLA group were elevated compared with the CON group (p < .05). In addition, ALA (600 mg/kg) treatment exhibited a reduced level of serum AST and TG (p < .05). Dietary ALA increased the activity of hepatic lipase in liver (p < .05). Supplemental 600 mg/kg ALA also improved the SOD activity and total antioxidant capacity level, along with a decreased MDA in ovarian tissue (p < .05). Furthermore, the mRNA expressions of ESR1, ESR2, VTG2 and ApoB in the liver and FSHR in follicles were upregulated in the HLA group (p < .05). In conclusion, dietary supplementation with 600 mg/kg ALA during the late egg-laying period could improve lipid metabolism and reproductive performance of breeder hens.

Mesenchymal Stem Cell Therapy Using Human Umbilical Cord in a Rat Model of Autoimmune-Induced Premature Ovarian Failure.

Premature ovarian failure (POF) is one of the principal causes of female infertility, and although its causes are complex and diverse, autoimmune deficiency may be involved. Human umbilical cord mesenchymal stem cells (UCMSCs) can be used for tissue regeneration and repair. Therefore, the present study was designed to determine the role of UCMSCs in immune factor-induced POF in rats. In this study, different concentrations of UCMSCs were injected into induced POF rats. Ovarian functions were examined by evaluating the estrus cycle, follicular morphology, hormonal secretion, and the proliferation and apoptosis of granulosa cells. Our results showed that the estrus cycle of rats returned to normal and follicular development was significantly improved after transplantation of UCMSCs. In addition, serum concentrations of 17-estradiol (E2), progesterone (P4), and anti-Müllerian hormone (AMH) increased significantly with treatment. Transplantation of UCMSCs also reduced the apoptosis of granulosa cells and promoted the proliferation of granulosa cells. All of these improvements were dose dependent. Furthermore, the results of related gene expression showed that transplanted human UCMSCs upregulated the expression of Bcl-2, AMH, and FSHR in the ovary of POF rats and downregulated the expression of caspase-3. These results further validated the potential mechanisms of promoting the release of cell growth factors and enhancing tissue regeneration and provide a theoretical basis for the clinical application of stem cells in the treatment of premature ovarian failure.

Dietary genistein supplementation protects against lipopolysaccharide-induced intestinal injury through altering transcriptomic profile.

Genistein is abundant in the corn-soybean meal feed. Little information is available about the effect of dietary genistein on the intestinal transcriptome of chicks, especially when suffering from intestinal injury. In this study, 180 one-day-old male ROSS 308 broiler chickens were randomly allocated to 3 groups, with 4 replicates (cages) of 15 birds each. The treatments were as follows: chicks received a basal diet (CON), a basal diet and underwent lipopolysaccharide-challenge (LPS), or a basal diet supplemented with 40 mg/kg genistein and underwent LPS-challenge (GEN). LPS injection induced intestinal injury and inflammatory reactions in the chicks. Transcriptomic analysis identified 7,131 differently expressed genes (3,281 upregulated and 3,851 downregulated) in the GEN group compared with the LPS group (P adjusted value < 0.05, |fold change| > 1.5), which revealed that dietary genistein exposure altered the gene expression profile and signaling pathways in the ileum of LPS-treated chicks. Furthermore, dietary genistein improved intestinal morphology, mucosal immune function, tight junction, antioxidant activity, apoptotic process, and growth performance, which were adversely damaged by LPS injection. Therefore, adding genistein into the diet of chicks can alter RNA expression profile and ameliorate intestinal injury in LPS-challenged chicks, thereby improving the growth performance of chicks with intestinal injury.

Oral Exposure to Genistein during Conception and Lactation Period Affects the Testicular Development of Male Offspring Mice.

Sexual hormones are essential for the process of spermatogenesis in the testis. However, the effect of maternal genistein (GEN) on the pups' testicular development remain-unclear. Our present study evaluated the effects of supplementing GEN for parental and offspring mice on the reproductive function and growth performance of the male pups. Mothers during gestation and lactation period were assigned to a control diet (CON group), low dose GEN (LGE group) diet (control diet +40 mg/kg GEN), and high dose of GEN (HGE group) diet (control diet +800 mg/kg GEN). Their male offspring underwent the same treatment of GEN after weaning. LGE treatment (40 mg/kg GEN) significantly increased body weights (p < 0.001), testes weights (p < 0.05), diameters of seminiferous tubule (p < 0.001) and heights of seminiferous epithelium (p < 0.05) of offspring mice. LGE treatment also increased serum testosterone (T) levels and spermatogenesis scoring (p < 0.05). However, HGE treatment (800mg/kg GEN) significantly decreased body weights (p < 0.001), testes weights (p < 0.05) and testis sizes (p < 0.001). Furthermore, mRNA expressions of ESR2 (p < 0.05), CYP19A1 (p < 0.001), SOX9 (p < 0.001) and BRD7 (p < 0.001) in testis of mice were increased in the LGE group. Similarly, HGE treatment increased mRNA expressions of ESR2 (p < 0.05) and CYP19A1 (p < 0.001). However, mRNA expressions of SOX9 and BRD7 were decreased significantly in the HGE group (p < 0.001). Meanwhile, higher ratio apoptotic germ cells and abnormal sperms were detected in the HGE group (p < 0.001). In conclusion, exposure to a low dose of GEN during fetal and neonatal life could improve testicular development of offspring mice, whereas, unfavorable adverse effects were induced by a high dose of GEN.

RNA Expression Profile and Alternative Splicing Signatures of Genistein-Treated Breeder Hens Revealed by Hepatic Transcriptomic Analysis.

Little information has been available about the influence of dietary genistein (GEN) on hepatic transcriptome of laying broiler breeder (LBB) hens. The study is aimed at broadening the understanding of RNA expression profiles and alternative splicing (AS) signatures of GEN-treated breeder hens and thereby improving laying performance and immune function of hens during the late egg-laying period. 720 LBB hens were randomly allocated into three groups with supplemental dietary GEN doses (0, 40 mg/kg, and 400 mg/kg). Each treatment has 8 replicates of 30 birds. Dietary GEN enhanced the antioxidative capability of livers, along with the increased activities of glutathione peroxidase and catalase. Furthermore, it improved lipid metabolic status and apoptotic process in the liver of hens. 40 mg/kg dietary GEN had the better effects on improving immune function and laying performance. However, transcriptome data indicated that 400 mg/kg dietary GEN did negative regulation of hormone biosynthetic process. Also, it upregulated the expressions of EDA2R and CYR61 by the Cis regulation of neighbouring genes (lncRNA_XLOC_018890 and XLOC_024242), which might activate NF-κB and immune-related signaling pathway. Furthermore, dietary GEN induced AS events in the liver, which also enriched into immune and metabolic process. Therefore, the application of 40 mg/kg GEN in the diet of breeder hens during the late egg-laying period can improve lipid metabolism and immune function. We need to pay attention to the side-effects of high-dose GEN on the immune function.

Freeze-Dried Royal Jelly Proteins Enhanced the Testicular Development and Spermatogenesis in Pubescent Male Mice.

Spermatogenesis and hormones secretions are crucial endocrine and physiological process for maintaining the life. Royal Jelly (RJ) bioactive components are Major Royal Jelly Proteins (MRJPs), owing exceptional biological properties. However, the effects of RJ on pup's testicular development during neonatal and pubertal period exposure hasn't been adequately studied. The aim of the study was to detect neonatal sexual hormones concentration and histopathological changes on testicular development of the male progeny after oral exposure to freeze-dried RJ for 35 consecutive days. After mice give birth, male pups were collected together, separated by sex, and randomly standardized to seven (7) male pups per dam. Male pups were assigned to control diet (CON group), low dose RJ (L-RJ group) diet (control diet + 125 mg/kg/day RJ), moderate dose RJ (M-RJ group) diet (control diet + 250 mg/kg/day RJ) and high dose of RJ (H-RJ group) diet (control diet + 500 mg/kg/day RJ). After weaning, male pups were continuously fed with freeze-dried RJ until the age of PNDs 35. The results revealed that, oral M-RJ (250 mg/kg/day) administration significantly (p < 0.05) increased the testis weight, the diameter of seminiferous tubule and the height of seminiferous epithelium of offspring mice at PNDs 14. However, high-dose RJ (500 mg/kg/day) decreased the diameter of seminiferous tubule but increased the height of seminiferous epithelium of male offspring (p < 0.05) at the same time point. Furthermore, oral M-RJ treatment significantly (p < 0.05) increased the testis weight and spermatogenesis at PNDs 21. Whereas, oral H-RJ treatment significantly (p < 0.05) reduced the diameter of seminiferous tubule and the height of seminiferous epithelium at PNDs 21. At PNDs 35, oral M-RJ treatment increased the testis weight, the diameter of seminiferous tubule and the level of FSH. While, high-dose of RJ reduced testis weight and size (diameter of seminiferous tubule and height of seminiferous epithelium), ratio of apoptotic germ cells and incomplete spermatogenesis collectively. In addition, sexual hormone secretions (FSH, LH, E2) were decreased after RJs treatment (L-RJ, M-RJ, H-RJ) at PNDs 21 respectively. In conclusion, the results concluded that oral administration of low and moderate doses of RJ could enhance the development of testis at neonate period until pubescent, whereas unfavorable adverse effects induced by high dose of RJ might remain.

Distributions of polycyclic musk fragrance in wastewater treatment plant (WWTP) effluents and sludges in the United States.

The polycyclic musks, AHTN and HHCB are fragrance ingredients widely used in consumer products. A monitoring campaign was conducted and collected grab effluent and sludge samples at 40 wastewater treatment plants (WWTP) across the United States to understand their occurrence and statistical distribution in these matrices. AHTN concentration in effluent ranged from <0.05 µg/L (LOQ) to 0.44 µg/L with a mean and standard deviation of 0.18 ± 0.11 µg/L. HHCB concentrations in effluent ranged from 0.45 to 4.79 µg/L with a mean of 1.86 ± 1.01 µg/L. AHTN concentrations in sludge ranged from 0.65 to 15.0mg/kg dw (dry weight) with a mean and standard deviation being 3.69 ± 2.57 mg/kg dw, while HHCB sludge concentrations were between 4.1 and 91 mg/kg with a mean of 34.0 ± 23.1mg/kg dw. Measured concentrations of AHTN and HHCB were significantly correlated with each other in both effluent and sludge. The concentrations of HHCB in both effluent and sludge were approximately an order of magnitude higher than those for AHTN, consistent with 2011 usage levels. The highest measured effluent concentrations for both AHTN and HHCB were below their respective freshwater PNECs (predicted no effect concentrations), indicating a negligible risk to biological communities below WWTPs, even in the absence of upstream dilution. Moreover, the large number of effluents and sludges sampled provides a statistical distribution of loadings that can be used to develop more extensive probabilistic exposure assessments for WWTP mixing zones and sludge amended soils.

MicroRNA-23b inhibits enterovirus 71 replication through downregulation of EV71 VPl protein.

Enterovirus 71 (EV71) is one of the causative pathogens of hand-foot-and-mouth disease and effective antiviral agents and vaccines against this virus have, to date, not been available. MicroRNAs (miRNAs) are a recently discovered class of RNAs with the function of post-transcriptional gene expression regulation. It has been demonstrated that miRNAs play important roles in the complicated interaction network between virus and host, while few studies have explored the role of miRNAs in EV71 infection. A recent study showed that hsa-miR-23b was downregulated significantly in cell-infected viruses. To address this issue, biological software miRanda was first used to predict possible target sites of miR-23b at EV71 gene sequence, then to confirm it by luciferase assay. miR-23b mimics were transfected to verify its effects on infection of EV71. These results suggest that miR-23b and upregulation of miR-23b inhibited the replication of EV71 by targeting at EV71 3'UTR conserved sequence. Taken together, miR-23b could inhibit EV71 replication through downregulation of EV71 VPl protein. These results may enhance our understanding on the prevention and treatment of hand-foot-and-mouth disease caused by EV71 infection.

[Prevalent characteristics in children with hand-foot-mouth disease in the Kunming area in 2010].

OBJECTIVE: To study the prevalent characteristics in children with hand-foot-mouth disease (HFMD) in the Kunming area in 2010. METHODS: The clinical data of 13286 outpatient and inpatient children with HFMD in Kunming Children's Hospital between January and December, 2010, including 8 death cases, 715 serious cases and 12563 non-serious cases, were retrospectively studied. RESULTS: Human enterovirus was detected in 8200 children (61.72%). Children infected with EV71 and CoxA16 accounted for 29.49% (2418/8200) and 53.21% (4363/8200), respectively. Seventy-five children (0.91%) were found to have a mixed infection of the two viruses. Other types of human enterovirus were detected in 1344 children (16.39%). There were significant differences in the total positive rate of human enterovirus in the four quarters of the year (P<0.01). The total positive rate in the second quarter represented the highest proportion (71.56%), and the number of patients was also highest, accounting for 52.94% of the total number of patients in the whole year. EV71 infection was common in the serious case group while CoxA16 was found to be the main pathogen in the non-serious case group. Serious cases were common in children under three years old. In the positive EV71 cases, the viral load of EV71 was not statistically different between the death cases, serious and non-serious cases. CONCLUSIONS: In 2010, children with HFMD in the Kunming area were mainly infected with CoxA16. Serious cases of HFMD were more common in those who were infected with EV71, and the majority of serious infections were suffered by children who were less than three years old. The viral load was not associated with disease severity. The highest morbidity rate was in the second quarter of the year.

Chondrogenic differentiation of canine myoblasts induced by cartilage-derived morphogenetic protein-2 and transforming growth factor-ß1 in vitro.

The meniscus has limited ability to repair itself following injury. However, tissue engineering provides new means of meniscus repair. Myoblasts, which possess the potential of multi-directional differentiation, may be ideal seed cells in meniscus tissue engineering. Myoblasts from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated myoblasts from canines and induced chondrogenesis in order to establish a new experimental model of seed cells. Myoblasts were isolated and harvested from Beagle canines. To induce chondrogenesis, cartilage-derived morphogenetic protein-2 (CDMP-2) at different concentrations (10, 20, 50 and 100 ng/ml), transforming growth factor-ß1 (TGF-ß1; 10, 20, 30 and 50 ng/ml), and different concentrations of CDMP-2 (10, 20, 50 and 100 ng/ml) together with TGF-ß1 (20 ng/ml) were added to the cultured pellets. After 21 days of in vitro culture, chondrogenic differentiation was evaluated by histological and immunohistochemical techniques. The degree of gene expression was measured by quantitative RT-PCR. Based on the histological staining of glycosaminoglycan, using the toluidine blue dye-binding method, we found that CDMP-2 initiated chondrogenic differentiation of myoblasts, as did TGF-ß1. Furthermore, CDMP-2 conferred a stronger stimulatory effect than TGF-ß1. The combination of CDMP-2 and TGF-ß1 synergistically induced chondrogenesis of myoblasts. This synergistic chondrogenic effect of CDMP-2 together with TGF-ß1 was further confirmed by quantification of glycosaminoglycan using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific proteins collagen I and II. Canine myoblasts can be induced into chondrocytes by CDMP-2 and TGF-ß1 in vitro, suggesting that myoblasts are suitable as seed cells for meniscus tissue engineering.

[Biochemical screening and genetic diagnosis of thalassemia in children from Kunming].

OBJECTIVE: To investigate the types and frequency of gene mutations in children with thalassemia in Kunming, Yunan Province. METHODS: A biochemical screening for thalassemia was performed by testing RBC fragility, MCV and hemoglobin electrophoresis on 1338 children from Kunming, Yunnan Province. Genetic diagnosis was performed on the children with α-thalassemia by gap-PCR and on the children with ß-thalassemia by PCR-RDB. RESULTS: The positive rate of the biochemical screening for thalassemia was 11.36% (152 cases). The positive rate of genetic diagnosis was 8.59% (115 cases). Of the 115 cases, α-thalassemia was found in 43 cases, ß-thalassemia in 68 cases and α-combined-ß thalassemia in 4 cases.--SEA/αα accounted for 47%, -α4.2/αα accounted for 21%, and HbH disease accounted for 14%. Six genotypes were found in 68 cases of ß-thalassemia and the mutation frequency of ßE was the highest (32%), followed by CD41-42 (24%), CD17 (23%), IVS-II654 (10%), CD71-72 (10%), and -28 (1%). CONCLUSIONS: The frequency of gene mutations for thalassemia is high in children from Kunming, Yunnan Province. Premarital and prenatal screenings and genetic diagnosis for thalassemia should be carried out in this area.

[Genotyping of Plasmodium vivax in China-myanmar border by circumsporozoite protein].

By using a series of type-specific primers for Plasmodium vivax circumsporozoite protein (CSP) gene and nested PCR, genotyping was conducted for the specimens of Plasmodium vivax isolated from China-Myanmar border. In 174 isolates of P. vivax, four genotypes, namely, tropical zone family strain, temperate zone family strain, genotype-mixed infection and PV-II type, were identified each accounting for 54.6%, 35.6%, 6.9%, and 2.9%, respectively. The tropical zone family strain was dominant in the border area. There was no significant difference on the P.v CSP genotype constitution between Laza isolate of Myanmar and Tengchong isolate of Yunnan, China.