Reference gene selection for expression studies in the reproductive axis tissues of Magang geese at different reproductive stages under light treatment.

In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.

Emerging preclinical modulators developed for F508del-CFTR have the potential to be effective for ORKAMBI resistant processing mutants.

BACKGROUND: F508del is prototypical of Class 2 CFTR mutations associated with protein misprocessing and reduced function. Corrector compounds like lumacaftor partially rescue the processing defect of F508del-CFTR whereas potentiators like ivacaftor, enhance its channel activity once trafficked to the cell surface. We asked if emerging modulators developed for F508del-CFTR can rescue Class 2 mutations previously shown to be poorly responsive to lumacaftor and ivacaftor. METHODS: Rescue of mutant CFTRs by the correctors: AC1, AC2-1 or AC2-2 and the potentiator, AP2, was studied in HEK-293 cells and in primary human nasal epithelial (HNE) cultures, using a membrane potential assay and Ussing chamber, respectively. RESULTS: In HEK-293 cells, we found that a particular combination of corrector molecules (AC1 plus AC2-1) and a potentiator (AP2) was effective in rescuing both the misprocessing and reduced function of M1101K and G85E respectively. These findings were recapitulated in patient-derived nasal cultures, although another corrector combination, AC1 plus AC2-2 also improved misprocessing in these primary tissues. Interestingly, while this corrector combination only led to a modest increase in the abundance of mature N1303K-CFTR it did enable its functional expression in the presence of the potentiator, AP2, in part, because the nominal corrector, AC2-2 also exhibits potentiator activity. CONCLUSIONS: Strategic combinations of novel modulators can potentially rescue Class 2 mutants thought to be relatively unresponsive to lumacaftor and ivacaftor.

ORKAMBI-Mediated Rescue of Mucociliary Clearance in Cystic Fibrosis Primary Respiratory Cultures Is Enhanced by Arginine Uptake, Arginase Inhibition, and Promotion of Nitric Oxide Signaling to the Cystic Fibrosis Transmembrane Conductance Regulator Channel.

ORKAMBI, a combination of the corrector, lumacaftor, and the potentiator, ivacaftor, partially rescues the defective processing and anion channel activity conferred by the major cystic fibrosis-causing mutation, F508del, in in vitro studies. Clinically, the improvement in lung function after ORKAMBI treatment is modest and variable, prompting the search for complementary interventions. As our previous work identified a positive effect of arginine-dependent nitric oxide signaling on residual F508del-Cftr function in murine intestinal epithelium, we were prompted to determine whether strategies aimed at increasing arginine would enhance F508del-cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in patient-derived airway epithelia. Now, we show that the addition of arginine together with inhibition of intracellular arginase activity increased cytosolic nitric oxide and enhanced the rescue effect of ORKAMBI on F508del-CFTR-mediated chloride conductance at the cell surface of patient-derived bronchial and nasal epithelial cultures. Interestingly, arginine addition plus arginase inhibition also enhanced ORKAMBI-mediated increases in ciliary beat frequency and mucociliary movement, two in vitro CF phenotypes that are downstream of the channel defect. This work suggests that strategies to manipulate the arginine-nitric oxide pathway in combination with CFTR modulators may lead to improved clinical outcomes. SIGNIFICANCE STATEMENT: These proof-of-concept studies highlight the potential to boost the response to cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, lumacaftor and ivacaftor, in patient-derived airway tissues expressing the major CF-causing mutant, F508del-CFTR, by enhancing other regulatory pathways. In this case, we observed enhancement of pharmacologically rescued F508del-CFTR by arginine-dependent, nitric oxide signaling through inhibition of endogenous arginase activity.

[Effect of glutamine-supplemented enteral nutrition on the apoptosis of colonic mucosal cells in young rats with inflammatory bowl disease].

OBJECTIVE: To study the effect of glutamine-supplemented enteral nutrition in regulating the apoptosis of intestinal mucosal cells and promoting mucosal healing in young rats with inflammatory bowl disease (IBD). METHODS: A total of 80 male Sprague-Dawley rats aged 4-5 weeks were randomly divided into 4 groups: blank control, IBD model, short peptide, and short peptide+glutamine (n=20 each). The IBD model was prepared by a single colon perfusion of 3-nitrobenzene sulfonic acid. At 3 days after modeling, the rats in the short peptide group were fed with short peptide formula (100 mL/kg), and those in the short peptide+glutamine group were fed with short peptide formula (100 mL/kg) and glutamine (0.5 g/kg). The course of intervention was 1 week. General conditions were observed after the experiment and their intestinal mucosal tissue was obtained. Hematoxylin-eosin staining was used to observe the pathological change of the intestinal mucosa. RT-PCR was used to measure the expression of apoptosis-regulating genes (bax and bc1-2) and apoptotic signal transduction factors (Caspase-3 and Caspase-9) in the intestinal mucosa. Western blot was used to measure the expression of insulin-like growth factor-1 (IGF-1) in the colonic mucosa. RESULTS: The IBD model group had poorer general conditions than the other three groups (blank control, short peptide and short peptide+glutamine), and the short peptide+glutamine group had better general conditions than the IBD model and short peptide groups. The IBD model group had significantly higher mRNA expression of bax than the other three groups (P<0.05). There was no significant difference in the mRNA expression of bcl-2, Caspase-3 and Caspase-9 among the 4 groups (P>0.05). The short peptide group had a significantly higher level of IGF-1 than the short peptide+glutamine, blank control and IBD model groups (P<0.05). CONCLUSIONS: Glutamine-supplemented enteral nutrition can effectively improve the general nutritional status of young rats with IBD, but it is not better than exclusive enteral nutrition in inhibiting the apoptosis of colonic mucosal cells and stimulating the synthesis of IGF-1 in the intestinal mucosa.

Bacteriology and antibiotic sensitivity of tonsillar diseases in Chinese children.

Although many bacteriology studies on tonsillar diseases have been completed, only a few studies investigated bacteriology of tonsillar diseases in recent years, especially in Asian children population. The aim of our study is to elucidate the bacterial flora and antibiotic sensitivity of tonsillar diseases in Chinese children. A three-center study was performed on 2994 children with or without tonsillar diseases. We compared and analyzed differences of bacterial pathogens among recurrent tonsillitis, tonsillar hypertrophy and controls. We found that on the surface of tonsil, Staphylococcus aureus, Haemophilus influenzae and Streptococcus pneumoniae were noted in the order given in the recurrent tonsillitis (RT) group. In the tonsillar hypertrophy (TH) and control group, H. influenzae, S. aureus and S. pneumoniae were noted in the order given. For the core of tonsil, H. influenzae, S. aureus and ß-hemolytic streptococcus were noted in the order given in both RT and TH group. S. aureus and H. influenzae were the most prevalent types of bacteria present in cultures containing two strains in the RT and TH group, respectively. We also observed some differences in the types of bacteria in the surface and core between the recurrent tonsillitis and tonsillar hypertrophy groups. Our study provides recent bacteria distribution and antibiotic sensitivity for tonsillar diseases in Chinese children and will be helpful in the treatment of these diseases.

Pharmacological Regulation of In Situ Tissue Stem Cells Differentiation for Soft Tissue Calcification Treatment.

Calcification of soft tissues, such as heart valves and tendons, is a common clinical problem with limited therapeutics. Tissue specific stem/progenitor cells proliferate to repopulate injured tissues. But some of them become divergent to the direction of ossification in the local pathological microenvironment, thereby representing a cellular target for pharmacological approach. We observed that HIF-2alpha (encoded by EPAS1 inclined form) signaling is markedly activated within stem/progenitor cells recruited at calcified sites of diseased human tendons and heart valves. Proinflammatory microenvironment, rather than hypoxia, is correlated with HIF-2alpha activation and promoted osteochondrogenic differentiation of tendon stem/progenitor cells (TSPCs). Abnormal upregulation of HIF-2alpha served as a key switch to direct TSPCs differentiation into osteochondral-lineage rather than teno-lineage. Notably, Scleraxis (Scx), an essential tendon specific transcription factor, was suppressed on constitutive activation of HIF-2alpha and mediated the effect of HIF-2alpha on TSPCs fate decision. Moreover, pharmacological inhibition of HIF-2alpha with digoxin, which is a widely utilized drug, can efficiently inhibit calcification and enhance tenogenesis in vitro and in the Achilles's tendinopathy model. Taken together, these findings reveal the significant role of the tissue stem/progenitor cells fate decision and suggest that pharmacological regulation of HIF-2alpha function is a promising approach for soft tissue calcification treatment.

[Development of human embryonic stem cell model for toxicity evaluation].

The current international standard for toxicity screening of biomedical devices and materials recommend the use of immortalized cell lines because of their homogeneous morphologies and infinite proliferation which provide good reproducibility for in vitro cytotoxicity screening. However, most of the widely used immortalized cell lines are derived from animals and may not be representative of normal human cell behavior in vivo, in particular in terms of the cytotoxic and genotoxic response. Therefore, It is vital to develop a model for toxicity evaluation. In our studies, two Chinese human embryonic stem cell (hESC) lines as toxicity model were established. hESC derived tissue/organ cell model for tissue/organ specific toxicity evaluation were developed. The efficiency and accuracy of using hESC model for cytoxicity, embryotoxicity and genotoxicity evaluation were confirmed. The results indicated that hESCs might be good tools for toxicity testing and biosafety evaluation in vitro.

Fingerprint of Hedyotis diffusa Willd. by HPLC-MS.

A HPLC-MS fingerprint method has been developed based on the consistent chromatographic features of the major chemical constituents among 10 batches of Hedyotis diffusa Willd. Chromatographic separation was conducted on a Hypersil-Keystone Hypurity C(18) column using methanol:water:acetic acid as the mobile phase. Major compounds, including oleanolic acid, ursolic acid and ferulic acid, were analysed by HPLC-MS. Their analysis was ascertained by comparison with data derived from the standard compounds. The HPLC-MS fingerprint was successfully applied to analyse and differentiate samples from different geographical origins, or processing methods. H. diffusa was well distinguished from Hedyotis chrysotricha by HPLC-MS. Therefore the establishment of fingerprint of H. diffusa is critical in assessing and controlling its overall quality.

Comparative study of pulmonary responses to nano- and submicron-sized ferric oxide in rats.

Ferric oxide (Fe(2)O(3)) nanoparticles are of considerable interest for application in nanotechnology related fields. However, as iron being a highly redox-active transition metal, the safety of iron nanomaterials need to be further studied. In this study, the size, dose and time dependent of Fe(2)O(3) nanoparticle on pulmonary and coagulation system have been studied after intratracheal instillation. The Fe(2)O(3) nanoparticles with mean diameters of 22 and 280 nm, respectively, were intratracheally instilled to male Sprague Dawley rats at low (0.8 mg/kgbw) and high (20 mg/kgbw) doses. The toxic effects were monitored in the post-instilled 1, 7 and 30 days. Our results showed that the Fe(2)O(3) nanoparticle exposure could induce oxidative stress in lung. Alveolar macrophage (AM) over-loading of phagocytosed nanoparticle by high dose treatment had occurred, while the non-phagocytosed particles were found entering into alveolar epithelial in day 1 after exposure. Several inflammatory reactions including inflammatory and immune cells increase, clinical pathological changes: follicular hyperplasia, protein effusion, pulmonary capillary vessel hyperaemia and alveolar lipoproteinosis in lung were observed. The sustain burden of particles in AM and epithelium cells has caused lung emphysema and pro-sign of lung fibrosis. At the post-instilled day 30, the typical coagulation parameters, prothrombin time (PT) and activated partial thromboplastin time (APTT) in blood of low dose 22 nm-Fe(2)O(3) treated rats were significantly longer than the controls. We concluded that both of the two-sized Fe(2)O(3) particle intratracheal exposure could induce lung injury. Comparing with the submicron-sized Fe(2)O(3) particle, the nano-sized Fe(2)O(3) particle may increase microvascular permeability and cell lysis in lung epitheliums and disturb blood coagulation parameters significantly.

Subchronic toxicity studies of Radix Astragali extract in rats and dogs.

Radix Astragali extract (RAE) is obtained from Astragalus membranaceus. It consists of Astragalus polysaccharide and Astragalus membranaceus saponins. In the study, we observed the subchronic toxicity of RAE in Sprague-Dawley rats and beagle dogs to evaluate the safety dosage range in clinical application. These subjects were daily administered of RAE by intra-peritoneum or vein for three consecutive months. General index were observed such as food-intake, behavior, body weight, hematological parameters, etc. Body weight, the weight of principal organ and hematology index are normal in experimental groups and control groups. The hematological biochemistry examination and histopathology examination of experimental groups are similar to control groups. In conclusion, our studies clearly demonstrated that RAE was safe without any distinct toxicity and side effects, the safety dosage range is 5.7-39.9g/kg for rats and 2.85-19.95g/kg for beagle dogs, which is equal to 70 or 35 times of that of human (0.57g/kg, say, average BW 70kg), respectively.

[Clinical study on the treatment of male immune infertility by Huzhangdanshenyin].

OBJECTIVE: To investigate the efficacy of the Chinese medicine Huzhangdanshenyin on male immune infertility, and provide an effective therapy for this disease. METHODS: Ninety men with immune infertility were selected as the research subjects and randomly divided into two groups, 60 in the treatment group, treated by Huzhangdanshenyin, and the other 30 in the control, treated by prednisone, both for 3 months. The improvement of clinical symptoms, immunologic indexes (antisperm antibodies in serum and seminal plasma) and sperm indexes (semen liquefied duration, motility, viability, density and abnormal morphology rate) were observed and the results analyzed. RESULTS: The total antisperm antibody reversing ratio of the treatment group was higher than that of the control (P < 0.01), especially the serum antisperm antibody reversing ratio. There were significant differences in the clinical cure rate and total validity rate between the treatment group and the control (P < 0.01). After the treatment, the markers of the clinical symptoms (P < 0.01), especially the serum antisperm antibody reversing ratio. There were significant differences in the clinical cure rate and total validity rate between the treatment group and the control (P < 0.01). After the treatment, the markers of the clinical symptoms were lower (P < 0.01), and the improvement of the clinical symptoms was better in the treatment group than in the control (P < 0.01), especially the symptoms of pain in the back and knees, distending and bearing-down sensation of the perineum and testis, hypersexuality and topalgias. Compared with pre-treatment, sperm motility and viability of the treatment group significantly improved (P < 0.01), and so did sperm density (P < 0.05). However, there were no significant differences in sperm density, semen liquefied duration, abnormal morphology rate and pH (P > 0.05) before and after the treatment. CONCLUSION: The Chinese medicine Huzhangdanshenyin works more effectively than prednisone in the treatment of male immune infertility. It could improve the antisperm antibody reversing ratio, clinical symptoms and signs and ameliorate sperm indexes with no obvious advierse effects.

[The hypoglycemic effects of crude polysaccharides extract from Momordica charantia in mice].

OBJECTIVE: In order to study the hypoglycemic effects of crude polysaccharides extract from Momordica charantia in normal and diabetic mice. METHODS: Oral glucose tolerance test was carried out in 24 normal mice, CPS was orally administered in experiment group at 1 g/kg BW. 80 diabetic mice (type 1 and type 2 diabetic model) were divided into two groups randomly, CPS was administered at ad lib and fasted condition in experiment group at 1 g/kg BW, blood glucose was measured at different time. RESULTS: In normal mice, after administered glucose (3g/kgBW) the blood glucose level of experiment group at 0.5, 1 h was significantly lower than that of control group (16.4% and 16.5% lower than control group respectively, P < 0.05 and P < 0.01). In diabetic mice, the fasted and ad lib blood glucose after administering CPS orally 2h, 4h were lower than that of control group (P < 0.05 and P < 0.01 respectively). CONCLUSION: CPS can improve OGTT in normal mice, and has significant hypoglycemic effect in two types diabetic mice.