Transcriptional regulatory region and DNA methylation analysis of TNNI1 gene promoters in Gaoyou duck skeletal muscle (Anas platyrhynchos domestica).

1. The slow skeletal muscle troponin I (TNNI1) gene has been found to be specifically expressed in slow muscle fibres and plays an important role in muscle development. The aim of this study was to determine the active control area of duck TNNI1 and identify the potential cis-regulatory elements in the promoter. 2. In this study, the TNNI1 promoter was first cloned by genome walking and the sequences were analysed using bioinformatics software. Firefly luciferase reporter gene vectors, driven by a series of constructs with progressive deletions, were used to identify the core transcriptional regulatory region of the duck TNNI1 gene. The methylation status of the CpG island in the TNNI1 promoter was detected in skeletal muscle on embryonic days 21 and 27, by bisulphite sequencing PCR (BSP). 3. The results showed two CpG islands presented in the promoter region, with one of the CpG islands located in the core transcriptional regulatory region (-2078/-885 bp). The total methylation levels of the 14 CpG sites were not altered between breast and leg muscles on embryonic days 21 and 27. However, four CpG sites (loci of positions 4, 11, 13, and 14) showed dramatically different methylation levels between breast and leg muscles at embryonic days 21 and 27. Analysis showed that multiple CpG sites had a significant correlation between the methylation levels of the CpG sites and mRNA expressions in skeletal muscle. Multiple transcription factor binding sites including Sp1, c-Myc, Oct-1 and NF-kB motifs were identified and might be responsible for transcriptional regulation of the TNNI1 gene. 4. These findings contribute to further understanding of the fundamental mechanism for transcriptional regulation of the TNNI1 gene in ducks.

Effect of bovine lactoferrin and human lactoferrin on the proliferative activity of the osteoblast cell line MC3T3-E1 in vitro.

We conducted a comparative in vitro study on the proliferative effects of natural human lactoferrin (nhLF) and bovine lactoferrin (bLF) on osteoblasts. We investigated cell proliferation, cell survival, cell cycle, and mRNA and protein expression of proliferating cell nuclear antigen. Results indicated that treatment with 100 µg/mL of bLF or nhLF promoted the proliferation and sustenance of osteoblasts, and increased the length of the G2/M and S phases compared with the untreated osteoblasts. Results of real-time quantitative PCR and Western blot showed that mRNA and protein expression of proliferating cell nuclear antigen by osteoblasts treated with bLF or nhLF were greater than those of the untreated control. At the same concentration, bLF demonstrated a greater effect on osteoblast proliferation than did nhLF. This study provides insights of significance in the utlization of bLF in healthy food formulas.

Genetic differences in ChTLR15 gene polymorphism and expression involved in Salmonella enterica natural and artificial infection respectively, of Chinese native chicken breeds, with a focus on sexual dimorphism.

Chicken Toll-like receptor 15 (ChTLR15) has been shown to participate in immune activation in response to various pathogens and in the innate defence against infection. Two genetically distinct Chinese breeds of chicken (Qinyuan Partridge and Baier breeds) were used to study the correlation between ChTLR15 single nucleotide polymorphisms and the natural infection status of salmonella in hens, and also to examine genetic and sex-specific effects on ChTLR15 mRNA expression in heterophils and spleen during acute infection with Salmonella enterica serovar Enteritidis (SE) from 1 to 10 days after experimental infection. Three single-nucleotide polymorphisms (G168A, C726T and A1166G) in a single exon of ChTLR15 were identified in the two breeds, but only C726T showed a significant association with salmonella infection. Compared with layer-type Baier chicks, meat-type Qingyuan chicks showed a higher tolerance for capture stress and (SE) infection, as measured, respectively, by the modified body weight of chicks in the control group and in the infection group. Meanwhile, ChTLR15 down-regulation in heterophils and up-regulation in spleen were involved in the response to pathogenic SE colonization during the acute infection period. These significant genetic effects in females led to greater differences in both innate and adaptive immune responses than those exhibited in males. These results suggest that genetics, time and gender play important roles in the modulation of ChTLR15 mRNA level elicited by the SE-mediated immune response differentially in the two genetically distinct breeds, with a focus on sexual dimorphism.

Analysis of the genetic effects of CAPN1 gene polymorphisms on chicken meat tenderness.

The micromolar calcium-activated neutral protease gene (CAPN1) is a physiological candidate gene for meat tenderness. Four previously identified single nucleotide polymorphism (SNP) markers located within the CAPN1 gene were evaluated for their associations with variation in the meat tenderness of a Chinese indigenous chicken breed, a higher meat quality breed (i.e., Qingyuan partridge chicken), and the commercial Recessive White chicken breed. Warner-Bratzler shear force measurements were used to determine tenderness phenotypes for all animals; intramuscular fat (IMF) content and rate of water loss in the breast muscles were also measured. Genotyping was performed by the polymerase chain reaction-ligase detection reaction method. Polymorphisms were identified for all markers, except CAPN1 2546. The frequency of allele T was zero, and allele C was fixed for CAPN1 2546 in the studied populations. The SNP CAPN1 3535 in the CAPN1 gene was significantly associated with tenderness and other meat quality traits, where animals inheriting the AA genotype had smaller shear force values, lower water loss rates, and higher IMF contents. Moreover, H1 (AAA) was the most advantageous haplotype for meat tenderness. The results of this study confirm some previously documented associations. Furthermore, novel associations have been identified that, following validation in other populations, could be incorporated into breeding programs to improve meat quality.

Upregulation of NRAMP1 mRNA confirms its role in enhanced host immunity in post-artificial infections of Salmonella enteritidis in chicks.

1. Salmonella enteritidis (SE) is reported as the most common food-borne pathogen transmitted through poultry products. The natural resistance-associated macrophage protein 1 (NRAMP1) is a candidate gene associated with SE-mediated immune response and is related to the phagocytosis of SE. In this study, the classical single-nucleotide polymorphism (SNP) G2357A in exon 8 of the NRAMP1 gene was detected. The expression of NRAMP1 mRNA was first investigated in heterophil granulocytes and spleen in chicks from two different Chinese native breeds at 1, 3 and 10 d post-infection. In addition, the association with the effect of SE challenge was identified. 2. The G2357A SNP showed no significant association with Salmonella natural infection in birds from two different Chinese native breeds. 3. The upregulation of NRAMP1 mRNA in heterophils and spleen was involved in the response to pathogenic SE colonisation during the acute infection period in chicks. The results suggest that genetics, age, gender and interactions among these factors play important roles in the modulation of NRAMP1 mRNA expression and copy number by SE-mediated immune response in different Chinese chickens. 4. In conclusion, the enhancement of host immunity mediated by the upregulation of NRAMP1 mRNA in heterophil granulocytes and spleen might be more obvious and earlier in the chicks resistant to infections with SE than in susceptible chicks.

Transcriptional co-activator PGC-1α gene is associated with chicken skeletal muscle fiber types.

The peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α) is a candidate gene for meat quality traits because of its prominent role in muscle fiber type switching and determination. We investigated the effects of the PGC-1α gene on chicken skeletal muscle fiber type switching and on other meat quality traits. Single nucleotide polymorphisms were detected by PCR-SSCP and DNA sequencing, and then genotyping was performed by PCR-ligation detection reaction methods. Skeletal muscle fiber types, intramuscular fat content, shear forces, and water loss rate of the gastrocnemius lateralis muscle were measured in Qingyuan Partridge chickens and Recessive White chickens. Four SNPs, C171T in exon2, C384T in exon3, G646A in exon5, and A948G in exon8 were detected. Marker-trait association analysis indicated that G646A polymorphism was associated with skeletal myofiber type and that H1 (CCAA) was the most advantageous haplotype for skeletal myofiber type. We concluded that polymorphisms of the PGC-1α gene and their haplotypes are associated with chicken skeletal myofiber type traits.

Association of chicken growth hormone polymorphisms with egg production.

Growth hormone (GH) has diverse functions in animals, together with other hormones from the somatotropic axis. Here, chicken GH (cGH) was investigated in recessive white chickens and Qingyuan partridge chickens as a candidate gene affecting egg production traits. Chicken egg production traits were studied in association with 4 selected single nucleotide polymorphisms (T185G, G662A, T3094C, and C3199T). Genotyping was performed by the polymerase chain reaction-ligase detection reaction method. T185G was significantly associated with the egg production traits of body weight at first egg (BW), egg weight at first egg (EW), and the total egg production of 300-day old birds (EN 300). T3094C was also significantly associated with certain egg production traits; however, it affected the 2 breeds differently. Haplotypes of the 4 single nucleotide polymorphisms were also significantly associated with egg production traits of chicken age at first egg laying, BW, EW, and EN 300. H1H6 was the most advantageous diplotype for egg production. We putatively concluded that polymorphisms in the cGH gene and its haplotypes could be used as potential molecular markers for egg production traits to enhance the breeding programs of indigenous chickens.

[Ultrasound-guided percutaneous cannulation for extracorporeal membrane oxygenation in children].

Objective: To evaluate the effectiveness and safety of ultrasound-guided percutaneous cannulation for extracorporeal membrane oxygenation (ECMO) in children. Methods: In this retrospective observational study, 66 cases who underwent non-cardiac surgery ECMO in pediatric intensive care unit (PICU) of Shanghai Children's Hospital from May 2016 to April 2021 were collected. The demographics, model of ECMO support, type and size of arteriovenous cannulas, way of catheterization and complications were recorded and summarized. Patients were divided into percutaneous cannulation group and surgical cannulation group according to catheterization strategies. The demographics, duration of cannulation and ECMO support, ECMO weaning rate and hospital survival rate were compared among two groups. χ2 and nonparametric rank sum test were used for comparison. Results: Among the 66 patients who received ECMO, 38 were male and 28 were female, with age 44.5 (12.0, 83.5) months and weight 15.0 (10.0, 25.0) kg; 21 patients underwent percutaneous cannulation, with a success rate of 95% (20 cases). Point-of-care ultrasound was performed for all percutaneous cannulation cases. The duration of percutaneous cannulation was significantly shorter than that of surgical cannulation (26.0 (23.3, 30.3) vs. 57.0 (53.8, 64.0) min, Z=6.31, P<0.001). Successful percutaneous cannulation cases were aged 70.5 (23.8, 109.5) months, and their weight was 23.2 (13.6, 37.0) kg. Ten cases were initially given veno-venous (VV) ECMO support, and 10 cases were given veno-arterial (VA) ECMO support. ECMO arterial cannulas were sized from 8 F to 17 F, and venous cannulas sized from 10 F to 19 F. For VV-ECMO, the right internal jugular and femoral veins were used as vascular access, while VA-ECMO used right internal jugular vein-femoral artery or right femoral vein-left femoral artery approach. Only one patient suffered severe complication (superior vena cava perforation). There was no catheter-related bloodstream infection. Conclusion: Ultrasound-guided percutaneous cannulation for ECMO can be performed with a high rate of success and safety in children.

[Analysis of risk factors of central nervous system complications supported on extracorporeal membrane oxygenation].

Objective: To investigate the risk factors of central nervous system (CNS) complications in children undergoing extracorporeal membrane oxygenation (ECMO) support. Methods: The clinical data, ECMO parameters, laboratory examination and outcome (follow-up to 90 d after discharge) of 82 children treated with ECMO in the pediatric intensive care unit (PICU) of Shanghai Children's Hospital from December 2015 to December 2021 were analyzed retrospectively in this study. The patients were divided into CNS complication group and non-CNS complication group. The ECMO mode, ECMO catheterization mode, clinical and laboratory indicators pre-ECMO and 24 h after ECMO initiation, in-hospital mortality and 90-day mortality were compared with Chi-square test, t test and nonparametric rank sum test. Kaplan-Meier method was used to draw survival curve, and Log-rank test was used to compare the difference in survival rate. The receiver operating characteristic (ROC) curve was used to evaluate the power of variables to predict CNS complications. Results: A total of 82 children were treated with ECMO, including 49 males and 33 females, aged 34 (8, 80) months. There were 18 cases suffering CNS complications, including cerebral hemorrhage in 8 cases, epilepsy in 6 cases, simple cerebral infarction in 3 cases, and cerebral hemorrhage combined with cerebral infarction in 1 case. Veno-arterial ECMO accounted for a greater proportion in CNS complication group (17/18 vs. 67% (43/64), χ2=4.02, P=0.045). A higher percentage of children with CNS complications underwent surgical cannulation compared to those in non-CNS complication group (16/18 vs. 53% (34/64), χ2=7.55, P=0.006). The laboratory results indicated that lower pre-ECMO pH value (7.24 (7.15, 7.28) vs. 7.35 (7.26, 7.45), Z=-3.65, P<0.001) and platelet count 24 h after ECMO initiation (66 (27, 135) ×109/L vs. 107 (61, 157) ×109/L, Z=-2.04, P=0.041) were associated with CNS complications. In the CNS complication group, 7 children died during hospitalization and 7 died during 90-day after admission, and there was no significant difference compared with those in the non-CNS complication group (7/18 vs. 31% (20/64), 7/18 vs. 34% (22/64), both P>0.05). The ROC curve analysis indicated that the area under the ROC curve for pre-ECMO pH value was 0.738 (95%CI 0.598-0.877), and the optimal cut-off value was 7.325. Conclusions: CNS complications in children undergoing ECMO support are common. Pre-ECMO pH value <7.325 is a risk factor for CNS complications. Reducing the veno-arterial ECMO and surgical cannulation can help reduce the occurrence of CNS complications.

Active pharmaceutical ingredients and mechanisms underlying phasic myometrial contractions stimulated with the saponin extract from Paris polyphylla Sm. var. yunnanensis used for abnormal uterine bleeding.

BACKGROUND: Total steroidal saponins of Paris polyphylla Sm. var. yunnanensis (TSSP) have been widely used in China for the treatment of abnormal uterine bleeding (AUB). But until now, the main active constituents and the mechanisms underlying the pharmacological actions on uterine activity have not been described. METHODS: Total steroidal saponins were extracted with EtOH and purified by chromatography. In vitro isometric contraction studies were performed using myometrial strips from estrogen-primed or pregnant rats. Intracellular calcium was monitored under a confocal microscope using Fluo-3 AM-loaded myometrial cells. RESULTS: TSSP dose-dependently induced phasic myometrial contractions in vitro. Experiments with calcium channel blockers or kinase inhibitors demonstrated that the TSSP-stimulated myometrial contraction was mediated by an increase in [Ca(2+)](i) via influx of extracellular calcium and release of intracellular calcium. Through bioassay-guided separation, it was found that total spirostanol saponins exhibited contractile activity in myometrium and Pennogenin-3-O-alpha-L-arabinofuranosyl(1-->4)[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (PARG) was identified as the active ingredient of TSSP. Furthermore, the contractile response of rat myometrium to PARG was significantly enhanced with advancing pregnancy. CONCLUSIONS: These data provide evidence that myometrial contractility stimulated by TSSP results from [Ca(2+)](i) increase and supports the possibility that some spirostanol gylcosides may represent a new type of contractile agonist for the uterus.

[Relationship between continuous renal replacement therapy and hypophosphatemia in critically ill children].

Objective: To investigate the incidence and prognosis of hypophosphatemia in critically ill children treated with continuous blood purification (CBP). Methods: The medical records of the critically ill patients, who were treated with CBP, admitted to pediatric intensive care unit (PICU) of Shanghai Children's Hospital from May 2014 to April 2017 were retrospectively analyzed. The serum phosphorus levels were tested before CBP, at 48-72 h during CBP, at the end of CBP and on the next day after CBP finished. Phosphorus supplement was given to the children with severe hypophosphatemia. Results: A total of 85 patients met the inclusion criteria. The serum phosphorus levels at the 4 indicated time points were (1.4±0.5), (0.7±0.3), (0.8±0.3), (0.9±0.4) mmol/L, respectively (F=45.21, P<0.05). Among the children, 66 cases (78%) had hypophosphatemia during CBP. The incidences of moderate and severe hypophosphatemia were 32 (48%) and 9 (14%), respectively. There were 41 patients with CBP replacement rates of (35-49) ml/(kg·h), while 44 patients with CBP replacement rates of 50-70 ml/(kg·h). There were significant differences at 48-72 h during CBP, the end of CBP and on the next day after CBP ((0.8±0.4) vs. (0.5±0.2), (1.0±0.3) vs. (0.6±0.2), and (1.1±0.4) vs. (0.8±0.2) mmol/L; t=7.672, 4.060, 14.440, P<0.05). Atotal of 9 cases were treated with sodium glycerophosphate. Among the 85 children, 24 (28%) patients died while 61 (72%) survived. There were no significant differences between the two groups in serum phosphorus levels at the indicated time points ((1.4±0.5) vs. (1.4±0.5), (0.7±0.3) vs. (0.7±0.3), (0.7±0.3) vs. (0.8±0.3), and (1.0±0.3) vs. (0.9±0.3) mmol/L, respectively, P>0.05). Conclusions: Hypophosphatemia is prone to occur during CBP, which probably related to the replacement rate. There was no significant relationship between hypophosphatemia and mortality in critically ill children after giving phosphorus supplementation.

Inhibitory effects of RRR-alpha-tocopheryl succinate on benzo(a)pyrene (B(a)P)-induced forestomach carcinogenesis in female mice.

AIM: To study the inhibitory effects of VES (RRR-alpha-tocopheryl Succinate, VES),a derivative of natural Vitamin E, on benzo(a)pyrene(B(a)P)-induced forestomach tumor in female mice. METHODS: The model of B(a)P-induced forestomach tumor was established according to the methods of Wattenberg with slight modify-cations. One hundred and eighty female mice (6 weeks old) were divided into six groups equally; negative control (Succinic acid), vehicle control (Succinate+B(a)P),positive control(B(a)P), high VES(2.5 g/kg.b.w+B(a)P), low VES(1.25 g/kg.b.w+B(a)P)ig as well as VES by ip (20 mg/kg.b.w+B(a)P). Except the negative control group, the mice were administrated with B(a)P ig. and corresponding treatments for 4 weeks to study the anti-carcinogenetic effect of VES during the initiation period. The experiment lasted 29 weeks, in which the inhibitory effects of VES both on tumor incidence and tumor size were tested. RESULTS: The models of B(a)P-induced forestomach tumor in female mice were established successfully. Some were cauliflower-like, others looked like papilla, even a few were formed into the ulcer cavities. VES at 1.25 g/kg.b.w, 2.5 g/kg.b.w. by ig and 20 mg/kg.b.w. via ip could decrease the number of tumors per mouse (1.7 plus minus 0.41, 1.6 plus minus 0.34 and 1.1 +/- 0.43), being lower than that of B(a)P group (5.4 +/- 0.32, P<0.05). The tumor incidence was inhibited by 18.2%, 23.1% and 50.0%. VES at 1.25 g/kg.b.w., 2.5 g/kg.b.w. by ig and 20 mg/kg.b.w. via ip reduced the total volume of tumors per mouse (54.8 +/- 8.84, 28.4 8 +/- 8.32 and 23.9 8 +/- 16.05), being significantly lower than that of B(a)P group (150.2 8 +/- 20.93, P < 0.01). The inhibitory rates were 63.5%, 81.1% and 84.1%, respectively. CONCLUSION: VES has inhibitory effects on B(a)P-induced forestomach carcinogenesis in female mice, especially by ip and it may be a potential anti-cancer agent in vivo.