miR-460b-5p promotes proliferation and differentiation of chicken myoblasts and targets RBM19 gene.

The meat production of broilers is crucial to economic benefits of broiler industries, while the slaughter performance of broilers is directly determined by skeletal muscle development. Hence, the broiler breeding for growth traits shows a great importance. As a kind of small noncoding RNA, microRNA (miRNA) can regulate the expression of multiple genes and perform a wide range of regulation in organisms. Currently, more and more studies have confirmed that miRNAs are closely associated with skeletal muscle development of chickens. Based on our previous miR-seq analysis (accession number: PRJNA668199), miR-460b-5p was screened as one of the key miRNAs probably involved in the growth regulation of chickens. However, the regulatory effect of miR-460b-5p on the development of chicken skeletal muscles is still unclear. Therefore, miR-460b-5p was further used for functional validation at the cellular level in this study. The expression pattern of miR-460b-5p was investigated in proliferation and differentiation stages of chicken primary myoblasts. It was showed that the expression level of miR-460b-5p gradually decreased from the proliferation stage (GM 50%) to the lowest at 24 h of differentiation. As differentiation proceeded, miR-460b-5p expression increased significantly, reaching the highest and stabilizing at 72 h and 96 h of differentiation. Through mRNA quantitative analysis of proliferation marker genes, CCK-8 and Edu assays, miR-460b-5p was found to significantly facilitate the transition of myoblasts from G1 to S phase and promote chicken myoblast proliferation. mRNA and protein quantitative analysis of differentiation marker genes, as well as the indirect immunofluorescence results of myotubes, revealed that miR-460b-5p significantly stimulated myotube development and promote chicken myoblast differentiation. In addition, the target relationship was validated for miR-460b-5p according to the dual-luciferase reporter assay and mRNA quantitative analysis, which indicates that miR-460b-5p was able to regulate RBM19 expression by specifically binding to the 3' UTR of RBM19. In summary, miR-460b-5p has positive regulatory effects on the proliferation and differentiation of chicken myoblasts, and RBM19 is a target gene of miR-460b-5p.

Comparative Analysis of miRNA Expression Profiles in Skeletal Muscle of Bian Chickens at Different Embryonic Ages.

MicroRNAs (miRNAs) are widely involved in the growth and development of skeletal muscle through the negative regulation of target genes. In order to screen out the differentially expressed miRNAs (DEMs) associated with skeletal muscle development of Bian chickens at different embryonic ages, we used the leg muscles of fast-growing and slow-growing Bian chickens at the 14th and 20th embryonic ages (F14, F20, S14 and S20) for RNA-seq. A total of 836 known miRNAs were identified, and 121 novel miRNAs were predicted. In the F14 vs. F20 comparison group, 127 DEMs were screened, targeting a total of 2871 genes, with 61 miRNAs significantly upregulated and 66 miRNAs significantly downregulated. In the S14 vs. S20 comparison group, 131 DEMs were screened, targeting a total of 3236 genes, with 60 miRNAs significantly upregulated and 71 miRNAs significantly downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the predicted target genes were significantly enriched in 706 GO terms and 6 KEGG pathways in the F14 vs. F20 group and 677 GO terms and 5 KEGG pathways in the S14 vs. S20 group. According to the interaction network analysis, we screened five coexpressed DEMs (gga-miR-146a-3p, gga-miR-2954, gga-miR-34a-5p, gga-miR-1625-5p and gga-miR-18b-3p) with the highest connectivity degree with predicted target genes between the two comparison groups, and five hub genes (HSPA5, PKM2, Notch1, Notch2 and RBPJ) related to muscle development were obtained as well. Subsequently, we further identified nine DEMs (gga-let-7g-3p, gga-miR-490-3p, gga-miR-6660-3p, gga-miR-12223-5p, novel-miR-327, gga-miR-18a-5p, gga-miR-18b-5p, gga-miR-34a-5p and gga-miR-1677-3p) with a targeting relationship to the hub genes, suggesting that they may play important roles in the muscle development of Bian chickens. This study reveals the miRNA differences in skeletal muscle development between 14- and 20-day embryos of Bian chickens from fast- and slow-growing groups and provides a miRNA database for further studies on the molecular mechanisms of the skeletal muscle development in Bian chickens.

Optimization of Cultivation Conditions of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes) for the Highest Antioxidant Activity and Antioxidant Content.

Ganoderma lucidum is a famous medicinal mushroom that is rich in antioxidants. The content of antioxidant components of grains can be effectively improved by G. lucidum as the fermenting strain. Optimization of the solid-state fermentation medium and optimization of the fermentation conditions were studied. The optimal fermentation substrate combination of G. lucidum TS (GL-TS) was 46.79% buckwheat, 53.21% rice; the optimal fermentation substrate combination of G. lucidum Am (GL-Am) was 4.17% soybean, 95.83% rice. The optimal fermentation conditions of GL-TS and GL-Am were as follows: inoculum amounts of 4.5% and 7.5%, temperatures of 30°C and 32°C, medium moisture content of 70% for both media, material granularities of 0.212-0.355 mm and 0.500-0.710 mm, and optimal fermentation time of 12.0 d and 10.5 d, respectively. Results of the analysis of antioxidant components in the fermentation substrates indicated that the antioxidant components were rich in antioxidant varieties and high in content. The contents of the antioxidant components (triterpenoids, total polyphenols, reducing sugars, anthocyanins, superoxide dismutase, glutathione, vitamin C, and vitamin E) in the full-fermentation substrates were greater than those in the nonfennentation substrates (except for flavonoids in the full-fermentation substrates, which were less than in the nonfennentation substrates). Glutathione was the major antioxidant component in the fermentation substrates, and the glutathione content was the highest. Therefore, the fermentation substrates of G. lucidum can be used to make antioxidant foods. This research contributes to the foundation for developing antioxidant foods based on G. lucidum.

Construction of Radial Defect Models in Rabbits to Determine the Critical Size Defects.

Many studies aimed at investigating bone repair have been conducted through animal models in recent years. However, limitations do exist in these models due to varying regeneration potential among different animal species. Even using the same animal, big differences exist in the size of critical size defects (CSD) involving the same region. This study aimed to investigate the standardization of radial bone defect models in rabbits and further establish more reliable CSD data. A total of 40 6-month-old New Zealand white rabbits of clean grade totaling 80 radial bones were prepared for bone defect models, according to the principle of randomization. Five different sizes (1.0, 1.2, 1.4, 1.7 and 2.0 cm) of complete periosteal defects were introduced under anesthesia. At 12 weeks postoperatively, with the gradual increase in defect size, the grades of bone growth were significantly decreased in all 5 groups. X-ray, CT scans and H&E staining of the 1.4, 1.7, and 2.0-cm groups showed lower grades of bone growth than that of the 1.0 and 1.2-cm groups respectively (P < 0.05). Using rabbit radial defect model involving 6-month-old healthy New Zealand white rabbits, this study indicates that in order to be critical sized, defects must be greater than 1.4 cm.

Clinical anatomy and mechanical tensile properties of the rectus femoris tendon.

PURPOSE: We aimed to provide anatomical data and mechanical tensile properties for the rectus femoris tendon to determine if it is a feasible substitute for the anterior cruciate ligament during knee joint reconstruction. METHODS: The length and width of the quadriceps femoris tendon were measured from ten adult cadavers (20 knees; age =48±2 years). The anatomic features of the patellar insertion on the quadriceps femoris tendon were also documented. The rectus femoris tendon and anterior cruciate ligament were harvested from an additional five fresh specimens (10 knees; age =41±3 years). To minimize dehydration, each specimen was wrapped in saline-moistened paper towels and stored at -10°C. We imposed tensile stresses on a total of twenty samples in a sample-driven machine at 10 mm/min until the specimens failed. RESULTS: The inserted and discrete widths of the rectus femoris tendon were 3.20±0.33 and 1.28±0.25 cm, respectively. The length of the tendon tissue was 6.96±0.80 cm and the length of mixing zone was 3.81±0.53 cm. The average thickness of the upper pole of the patella was 2.22±0.14 cm. In mechanical tensile properties, the unit modulus and unit maximum load of the rectus femoris tendon were both 63% of the anterior cruciate ligament. CONCLUSIONS: Based on its anatomical and mechanical tensile properties, the rectus femoris tendon is a feasible donor site to reconstitute the anterior cruciate ligament.

Presepsin as a novel sepsis biomarker.

BACKGROUND: In 2004, a new biomarker sCD14-subtypes (presepsin) was found and its value was shown in the diagnosis and evaluation of sepsis. This article is a brief overview of the new biomarker. DATA SOURCES: A literature search using multiple databases was performed for articles, especially meta-analyses, systematic reviews, and randomized controlled trials. RESULTS: Compared with other markers, presepsin seems to have a better sensitivity and specificity in the diagnosis of sepsis. Presepsin as a biom1arker is not only suitable for the early diagnosis of sepsis, but also for the assessment of its severity and prognosis. CONCLUSIONS: Presepsin has a higher sensitivity and specificity in the diagnosis of sepsis as a new biomarker, and is a predictor for the prognosis of sepsis. More importantly, preseptin seems to play a crucial role as a supplemental method in the early diagnosis of sepsis. Since there is no multicenter study on the relationship between presepsin and sepsis, further studies on the clinical values of presepsin are needed.

Inhibition of SENP5 suppresses cell growth and promotes apoptosis in osteosarcoma cells.

SUMOylation is a dynamic and reversible process. Several SUMO-specific proteases (SENPs) that remove SUMO from substrates have been shown to be amplified in a subset of cancers. SENP5 is required for cell division, as well as maintaining mitochondrial morphology and function. SENP5 has been reported to be predominantly localized to the cytoplasm of oral squamous cell carcinoma (OSCC) and is associated with the differentiation of OSCC. Western blot was used to detect the protein expression of SENP5 in osteosarcoma cells and tissue. Lentivirus-mediated siRNA was used to silence the expression of SENP5. Cell cycle distribution was determined by FACS analysis. The present study showed that SENP5 is overexpressed in osteosarcoma cells. In addition, lentivirus-mediated small interfering RNA (siRNA) of SENP5 significantly inhibited cell growth and induced apoptosis in osteosarcoma cells. SENP5 inhibition suppressed the growth and colony formation capacity of two osteosarcoma cell lines, U2OS and Saos-2. Silencing the expression of SENP5 in serum-starved U2OS and Saos-2 cells induced an increase in caspase-3/-7 activity and a decrease in cyclin B1 expression. These observations indicate that SENP5 is required for cell growth and apoptosis and may therefore be a promising drug target for antiosteosarcoma treatment.

Predicting value of serum soluble ST2 and interleukin-33 for risk stratification and prognosis in patients with acute myocardial infarction.

BACKGROUND: Acute myocardial infarction (AMI) is a common cardiac emergency with high mortality. Serum soluble ST2 (sST2) is a new emerging biomarker of cardiac diseases. The present study is to investigate the predictive value of sST2 and interleukin-33 (IL-33) for risk stratification and prognosis in patients with AMI. METHODS: Fifty-nine patients with AMI, whose chief complaint was chest pain or dyspnea, were selected for our study. Physical examination, chest radiograph, electrocardiograph (ECG), biomarkers of myocardial infarction, NT-proBNP, echocardiography and other relevant examinations were performed to confirm the diagnosis of AMI. Thirty-six healthy people were chosen as the control group. Serum samples from these subjects (patients within 24 hours after acute attack) were collected and the levels of sST2 and IL-33 were assayed by enzyme-linked immuno-sorbent assay (ELISA) kit. The follow-up was performed on the 7th day, 28th day, 3rd month and 6th month after acute attack. According to the follow-up results we defined the end of observation as recurrence of AMI or any causes of death. RESULTS: Median sST2 level of the control group was 9.38 ng/ml and that of AMI patients was 29.06 ng/ml. Compared with the control group, sST2 expression in the AMI group was significantly different (P < 0.001). In contrast, the IL-33 level showed no significant difference between the two groups. Serum sST2 was a predictive factor independent of other variables and may provide complementary information to NT-proBNP or GRACE risk score. IL-33 had no relationship to recurrence of AMI. Both sST2 and the IL-33/sST2 ratio were correlated with the 6-month prognosis; areas under the ROC curve were 0.938 and 0.920 respectively. CONCLUSIONS: Early in the course (<24 hours) of AMI, sST2 usually increases markedly. The increase of sST2 has an independent predictive value for the prognosis in AMI patients and provides complementary information to NT-proBNP or GRACE risk score. The IL-33/sST2 ratio correlates with the 6-month prognosis of AMI patients. However, there is no significant relationship between IL-33 and the prognosis of AMI patients.

PPIase independent chaperone-like function of recombinant human Cyclophilin A during arginine kinase refolding.

Whether Cyclophilin A (CyPA) functions as a foldase or a chaperone when assisting protein folding has long been argued. In this study, we engineered four variants of recombinant human Cyclophilin A (rhCyPA), all of which were inactive to tetrapeptide substrate Suc-AAPF-pNA. However, these variants were able to suppress aggregation during arginine kinase (AK) refolding as efficient as wild-type rhCyPA, especially, variant Q63A had even more efficiency to suppress aggregation and improve reactivation yields of AK. These results indicate that rhCyPA have peptidyl-prolyl cis-trans isomerase (PPIase) independent chaperone-like activity during AK folding. In addition, results suggest that surface hydrophobicity of rhCyPA can suppress AK aggregation and binding to rhCyPA hydrophobic active pocket is a prerequisite for chaperoning AK folding.

Mutation of residue arginine 330 of arginine kinase results in the generation of the oxidized form more susceptible.

Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg(2+)ATP to form phosphoarginine and Mg(2+)ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK.

Clinical characteristics and prognosis of heart failure with normal left ventricular ejection fraction in elderly patients.

BACKGROUND: The term heart failure with normal ejection fraction (HFNEF) is often used to describe the syndrome of heart failure with normal ejection fraction. Based on the previous studies, HFNEF has a significant morbidity and mortality and is associated with a similar prognosis to heart failure with reduced ejection fraction (HFREF). The present study aimed to investigate the clinical characteristics and prognosis of HFNEF in elderly patients. METHODS: Consecutive elderly patients (≥ 60 years old) hospitalized for the first episode of heart failure (HF) in Beijing Hospital from January 2003 to December 2009 were retrospectively recruited. Three hundred and ten patients with HF were eligible for our study. As recently recommended, a cut-off value of 50% was used to distinguish HFNEF (LVEF ≥ 50%) from HFREF (LVEF < 50%). Data were retrospectively obtained from hospital records and databases. Follow-up data were obtained by telephone and from hospital records. For every eligible patient, the clinical characteristics and prognosis were collected and compared between the HFNEF and HFREF groups. RESULTS: Patients with HFNEF accounted for 54.5% of all cases of elderly patients with HF. Compared with HFREF, the elderly patients with HFNEF had a higher proportion of females (62.1% vs. 32.6%, P < 0.001), higher body mass index (BMI) ((24.9 ± 4.7) vs. (23.5 ± 4.0) kg/m(2), P = 0.011), higher systolic blood pressure at admission ((141.5 ± 22.6) vs. (134.3 ± 18.6) mmHg, P = 0.002), but lower hemoglobin levels ((118.3 ± 22.7) vs. (125.8 ± 23.8) g/L, P = 0.005). The incidence of coronary heart disease (43.2% vs. 65.2%, P < 0.001) and myocardial infarction (16.6% vs. 46.1%, P < 0.001) were significantly lower in elderly patients with HFNEF than in those with HFREF (P < 0.001). With a mean follow-up of 33.5 (0.5 - 93) months, 120 patients (38.7%) died, including 94 (30.3%) cardiac deaths. The HFNEF group had fewer deaths than the HFREF group at the end of the first follow-up (46/169 (27.2%) vs. 58/141 (41.1%)) and at the end of the second follow-up (56/169 (33.1%) vs. 64/141 (45.4%)). Kaplan-Meier survival analysis showed a significantly higher survival rate in elderly patients with HFNEF than those with HFREF (P = 0.021 for total mortality and P < 0.001 for cardiac mortality). Multiple Logistic regression analysis showed that LVEF < 50% was an independent risk factor for death in elderly patients with HF. CONCLUSIONS: More than half of elderly patients with HF have a normal LVEF. The prognosis of the elderly patients with HFNEF is poor, though slightly better than the elderly patients with HFREF.

Evidences of monomer, dimer and trimer of recombinant human cyclophilin A.

Cyclophilin A (CyPA) is a cytosolic receptor of immunosuppressive drug cyclosporin A (CsA) which possesses peptidyl-prodyl cis/trans isomerase (PPIase) activity. The recombinant human CyPA (rhCyPA) gene has been expressed in E. coli M15. Purification was performed using salting-out, as well as Sephacryl S-100 and DEAE-Sepharose CL-6B column chromatography. The molecular weight is about 18 kDa, confirmed by SDS-PAGE and mass spectrum. The results of Native-PAGE and immunoblotting showed the existence of three bands, which agreed well with the gel filtration results. The molecular mass of the three bands determined via CTAB gel electrophoresis and SDS-PAGE (rhCyPA cross-linked with glutaraldehyde) was 18 kDa, 36 kDa and 54 kDa respectively. Further more, the native rhCyPA and the cross-linked rhCyPA had the similar chromatographic behavior in gel filtration. All of the evidences above suggest that rhCyPA exists in forms of monomer, dimer and trimer. Moreover, we observed that even at low protein concentrations CyPA largely occurs as a dimer in solution, and enzyme kinetic parameters showed that activity of dimer was much higher than monomer or trimer, which probably have some biological significances.

[Risk factors and warning signs of sudden cardiac death].

Sudden cardiac death (SCD) is the most common death mode. No test has been able to accurately predict SCD. This article reviews the risk factors and warning signs of SCD with an attempt to effectively prevent SCD events.