Clusterin suppresses spermatogenic cell apoptosis to alleviate diabetes-induced testicular damage by inhibiting autophagy via the PI3K/AKT/mTOR axis.

BACKGROUND INFORMATION: Diabetes-induced testicular dysfunction is characterised by abnormal apoptosis of spermatogenic cells, but the underlying mechanism is poorly understood. This study aimed to investigate the roles of clusterin (CLU) in testicular damage associated with diabetes pathogenesis, as well as the molecular mechanism. A rat diabetes model was established using streptozocin, and the mouse spermatogenic cell line GC-1 spg was treated with high glucose as a cellular model. CLU was overexpressed in GC-1 spg cells, followed by detection of serum testosterone, cell proliferation, cell apoptosis and autophagy. RESULTS: CLU expression was significantly reduced and LC3 expression was elevated in testis tissues in the rat diabetes model and high glucose-treated GC-1 spg cells. High glucose led to suppressed viability, enhanced apoptosis, reduced Bcl-2 expression, elevated Bax expression and cleavage of Caspase-3/-9 in GC-1 spg cells, and these effects were abrogated by CLU overexpression. Additionally, CLU overexpression repressed LC3 and Beclin-1 expression, reduced the LC3II/LC3I ratio and promoted p62 expression in GC-1 spg cells in the presence of high glucose, and these effects were all mitigated by rapamycin treatment. Inhibition of PI3K/AKT/mTOR signalling with LY294002 activated autophagy in CLU-overexpressing GC-1 spg cells under high glucose conditions. CLU overexpression repressed autophagy and alleviated testicular damage in diabetic rats, which was also abrogated by LY294002 treatment. CONCLUSIONS: CLU expression is suppressed during diabetes-induced testicular damage, whereas CLU overexpression alleviates diabetes-induced testicular damage by activating PI3K/AKT/mTOR signalling to inhibit autophagy and further repress spermatogenic cell apoptosis.

Quantitative study of cellular heterogeneity in doxorubicin uptake and its pharmacological effect on cancer cells.

Cellular heterogeneity in doxorubicin (DOX) uptake and its relationship with pharmacological effect on cancer cells were quantitatively investigated for the first time. An in vitro experimental model was established by treating human leukemia K562 and breast cancer MCF-7 cells with different schedules of DOX with or without surface P-glycoprotein (P-gp) inhibitor verapamil (VER). The cellular heterogeneity in DOX uptake was quantitatively examined by single-cell analysis using capillary electrophoresis coupled with laser-induced fluorescence detection. The corresponding cytotoxic effect was tested by cellular morphology, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium and flow cytometry assays. The expression of cellular membrane surface P-gp was determined by flow cytometry. Results showed that the cellular heterogeneity exists in DOX uptake. The single-high DOX schedule leads to lower uptake heterogeneity and higher mean drug uptake. The cellular heterogeneity in DOX uptake was found to be negatively correlated with drug cytotoxicity and surface P-gp expression, with r = -0.7680 to ~ -0.9587. VER reduces the cellular variation in DOX uptake, suggesting that surface P-gp may be one of the causes of the cellular heterogeneity in DOX uptake. This research demonstrates the importance of quantitative study of cellular heterogeneity in drug uptake and its potential application in drug schedule design, response prediction and therapy modulation.

[The genetic features of drug resistance to group A streptococcus and macrolides antibiotics among pediatric patients in Beijing 2012].

OBJECTIVE: To investigate the genetic features of drug resistance to group A streptococcus(GAS) and macrolides antibiotics among pediatric patients in Beijing 2012. METHODS: A total of 199 strains of GAS were collected from 36 hospitals in Beijing between May and July, 2012. All strains were isolated from oropharyngeal swabs. The minimum inhibitory concentrations (MICs) of ten antibiotics (penicillin, ampicillin, erythromycin, clindamycin, tetracycline, levofloxacin, tigecycline, vancomycin, linezolid and streptogramin) were detected by VITEK-2 compact with GPS-67 test kit. The genes encoding macrolides resistance (ermA, ermB and mefA ) were amplified and tested by PCR. The macrolides resistant phenotype of group A streptococcus was detected by double disc test (D-test). RESULTS: Among 199 strains of GAS collected in this study, 101(50.8%) were from suburbs and the other 98(49.2%) were from urban areas. 111(55.8%) strains were collected from scarlet fever patients while the other 88(44.2%) were from oropharyngeal infection cases. All the strains were sensitive to penicillin and ampicillin, and the percentage of resistance to erythromycin, clindamycin and tetracycline were 96.5% (192/199), 95.5% (190/199) and 92.0% (183/199), respectively. All strains were susceptible to levofloxacin, tigecycline, vancomycin, linezolid and streptogramin. The rates of resistance to erythromycin, clindamycin and tetracycline were different in different districts, however, the difference in it between ages and clinical diagnosis did not show statistical significance (P > 0.05) . The detected rate of drug resistance gene ermB was 98.5% (196/199). The gene ermA was only detected out in 5 strains and the gene mefA was not detected out. 199 strains showed A macrolides resistant phenotype cMLS, while the phenotype iMLS was not found in this study. CONCLUSION: This study demonstrates the high level of clindamycin resistance in group A streptococcus collected from children in Beijing, 2012. The macrolides resistance of group A streptococcus was highly prevalent in Beijing, and the dominant phenotype was cMLS mediated by gene ermB.

[Application of EARS in early-warning of influenza pandemic in Beijing].

OBJECTIVE: To illustrate the efficiency of cumulative sum (CUSUM) in pre-warning of the influenza peak in Beijing. METHODS: CUSUM was used to analyze the data of influenza like illness (ILI), and the results of the influenza laboratory surveillance was regarded as the gold standard to judge the approaching of the influenza peak. RESULTS: The surveillance was launched in 421 hospitals in Beijing during the 2009 to 2010 influenza season, while the influenza laboratory surveillance was launched by 7 collaborative laboratories. From Jun. 2009 to Apr. 2010, the average ILI percentage in the 421 hospitals was 2.56%. In the study, 19 262 pharyngeal swab samples were collected from the ILI cases in 11 hospitals and 5 045 of them were tested positive for the influenza virus, with the novel swine-origin influenza A H1N1 virus dominating. After analyzing of the ILI surveillance data with CUSUM, it was found that the ILI surveillance in Beijing could make a satisfactory early warning for the approaching of the influenza peak referring to the gold standard based on the influenza laboratory results. CONCLUSION: It could give the prediction and early warning for the influenza peak efficiently and precisely, by using CUSUM to analyze the influenza surveillance data of Beijing.

Gestational Leucylation Suppresses Embryonic T-Box Transcription Factor 5 Signal and Causes Congenital Heart Disease.

Dysregulated maternal nutrition, such as vitamin deficiencies and excessive levels of glucose and fatty acids, increases the risk for congenital heart disease (CHD) in the offspring. However, the association between maternal amino-acid levels and CHD is unclear. Here, it is shown that increased leucine levels in maternal plasma during the first trimester are associated with elevated CHD risk in the offspring. High levels of maternal leucine increase embryonic lysine-leucylation (K-Leu), which is catalyzed by leucyl-tRNA synthetase (LARS). LARS preferentially binds to and catalyzes K-Leu modification of lysine 339 within T-box transcription factor TBX5, whereas SIRT3 removes K-Leu from TBX5. Reversible leucylation retains TBX5 in the cytoplasm and inhibits its transcriptional activity. Increasing embryonic K-Leu levels in high-leucine-diet fed or Sirt3 knockout mice causes CHD in the offspring. Targeting K-Leu using the leucine analogue leucinol can inhibit LARS activity, reverse TBX5 K-Leu modification, and decrease the occurrence of CHD in high-leucine-diet fed mice. This study reveals that increased maternal leucine levels increases CHD risk in the offspring through inhibition of embryonic TBX5 signaling, indicating that leucylation exerts teratogenic effects during heart development and may be an intervening target of CHD.

Effects of MCRS1 on proliferation, migration, invasion, and epithelial mesenchymal transition of gastric cancer cells by interacting with Pkmyt1 protein kinase.

Microspherule protein 1(MCRS1) is known to be an oncogene in several tumors. However, recent studies have shown that MCRS1 inhibits lymphatic metastasis in gastric cancer (GC) patients by inhibiting telomerase activity. Protein kinase, membrane associated tyrosine/threonine 1(Pkmyt1), a member of the WEE1 family, has been found to interact with MCRS1 by yeast two-hybrid assay; however, how these two proteins interact in GC is still unclear. Hence, this study aimed to investigate the effect of MCRS1 interaction with Pkmyt1 on GC cell proliferation, migration, and invasion. Initially, we observed increased expression of MCRS1 in GC SGC-7901 cells and decreased expression in GC BGC-823 cells. Hence, we down-regulated MCRS1 expression in SGC-7901 cells and up-regulated it in BGC-823 cells. Our results showed that overexpression of MCRS1 inhibits the growth, invasion and migration of GC cells, while downregulation of MCRS1 promotes the growth, invasion and migration of GC cells. When MK1775, an inhibitor of WEE1 kinase, was added after downregulation of MCRS1, phenotypic recovery effects were observed. Overexpression of MCRS1 also inhibited the expression of Pkmyt1 and vice versa. This indicated that there might be a possible interaction between MCRS1 and Pkmyt1. Furthermore, immunoprecipitation assay revealed the interaction between MCRS1 and Pkmyt1 in virto, and immunofluorescence experiments showed that the two proteins were co-localized in the cytoplasm. In conclusion, our study confirmed the specific tumor suppressive activity of MCRS1 in GC proliferation, invasion and migration and suggested that it might inhibit the progression of GC through its interaction with Pkmyt1.

A Mobile Sleep-Management Learning System for Improving Students' Sleeping Habits by Integrating a Self-Regulated Learning Strategy: Randomized Controlled Trial.

BACKGROUND: Insomnia can significantly affect students' learning performance. Researchers have indicated the importance and challenge of coping with insomnia using nondrug treatments, such as cognitive behavioral therapy (CBT) for insomnia. However, it is easy for the traditional CBT for insomnia to be interrupted owing to the overly lengthy period of sleep therapy. Self-regulated learning (SRL) strategies are known to be an effective approach for helping students improve their time management, as well as their ability to set learning goals and adopt learning strategies. OBJECTIVE: The objective of this study was to propose a mobile sleep-management learning system integrated with SRL strategies and CBT. METHODS: A total of 18 undergraduate students from a university in northern Taiwan participated in the 2-week experiment of using this sleep-management system. RESULTS: The experimental results showed that the proposed approach was useful and easy for students to use. In addition, the number of students with insomnia significantly decreased; that is, the proposed approach could help students improve their sleep quality and cultivate better sleeping habits, which is important for them to enhance their learning efficiency. CONCLUSIONS: With the assistance of this proposed approach, students can plan their daily life by setting goals, applying strategies, monitoring their life habits process, and modifying strategies to cultivate good learning and healthy lifestyle habits. TRIAL REGISTRATION: Government Research Bulletin MOST104-3011-E038-001; https://www.grb.gov.tw/search/planDetail? id=11568383&docId=467988 (Archived by WebCite at http://www.webcitation.org/73MnPHNri).

Elevated expression of nuclear protein kinase CK2α as a poor prognosis indicator in lymph node cancerous metastases of human thyroid cancers.

AIM: To investigate the expression of protein kinase CK2α (CK2α) in human thyroid disease and its relationship with thyroid cancer metastasis. MATERIALS AND METHODS: Using immunohistochemistry we measured the expression of CK2α in 76 benign and malignant human thyroid cancer tissues, including 10 pairs of papillary carcinoma tissues with or without lymph node cancerous metastasis and similarly 10 pairs of lymph nodes. RESULTS: The expression of CK2α was found to be higher in thyroid carcinoma cases (papillary carcinoma, follicular carcinoma, anaplastic carcinoma and medullary carcinoma) than in ones such as chronic lymphocytic thyroiditis, nodular goiter and adenoma. These findings were also confirmed by RT-PCR and Western blotting. More strikingly, elevated expression of CK2α in thyroid papillary carcinoma tissues was not only significantly associated with lymph node cancerous metastasis and clinical stage of thyroid cancers; but also correlated with epithelial-mesenchymal transition (EMT) and high tenascin C (TNC) expression. In addition, EMT and high TNC expression in thyroid carcinoma tissues was significantly associated with lymph node cancerous metastasis. CONCLUSIONS: Elevated expression of nuclear CK2α is a poor prognosis indicator in lymph node cancerous metastasis of human thyroid cancers.

[Characteristics and related factors related to the resistance on antibiotics among group A streptococcus strains isolated from children in Beijing, during May and July 2011].

OBJECTIVE: To analyze the characteristics of antibiotic resistance on group A streptococcus isolated from pediatrics in Beijing in 2011, to provide reference for clinical drug administration. METHODS: Strains of group A streptococcus were collected from the Departments of Pediatrics in 36 hospitals at different Districts of Beijing, from May to July 2011. Minimal inhibitory concentrations (MIC) with ten antibiotics of these isolates were tested by VITEK 2 Compact method. All the Susceptibility rate (S%), Intermediate rate (I%) and Resistance rate (R%) were calculated according to their MIC values. The macrolides resistant phenotype of group A streptococcus was detected by D-test. RESULTS: A total of 633 (19.1%) group A streptococcus strains were cultured from 3315 throat swabs. All the isolates were susceptible to penicillin, ampicillin, streptogramin, linezolid, tigecycline, vancomycin, while 96.5% (611/633) of the isolates were susceptible to levofloxacin. A total of the 96.1% (608/633) isolates exhibited resistance to erythromycin. The resistance rates to clindamycin and tetracycline were 79.3% (502/633) and 93.7% (593/633), respectively. A total of 9 different resistant patterns were observed, with the dominant patterns as:concomitant resistance to erythromycin, clindamycin and tetracycline (72.7%, 460/633), followed by combined resistance to erythromycin and tetracycline (18.0%, 114/633). The most commonly seen macrolide resistant phenotype was cMLS type (83.2%). In total, 97 strains belonged to iMLS type and 5 strains to M type. Data through multivariate logistic regression analysis showed that factors as occupation and samples being collected from the sub-urban areas etc. were significantly associated with the resistance rates to tetracycline and the odds ratio (95%CI) as 2.43 (1.16 - 5.09) and 2.35 (1.47 - 3.73). Isolates collected from the sub-urban areas were significantly associated with resistance rates to clindamycin, with the odds ratio (95%CI) being 0.48 (0.25 - 0.92). CONCLUSION: All the isolates acquired from the Pediatrics Departments in Beijing were susceptible to penicillin and ampicillin. The high resistance rates of erythromycin, clindamycin and tetracycline resistance to group A streptococcus were observed, with the major resistant phenotype as cMLS. Factors as occupation and the collection site of samples were significantly associated with the resistance rates to tetracycline while the sites of sample collection were significantly associated with the resistance rates to clindamycin.

Rapamycin inhibits re-endothelialization after percutaneous coronary intervention by impeding the proliferation and migration of endothelial cells and inducing apoptosis of endothelial progenitor cells.

Endothelial-cell function is important in the healing of damaged endothelium after percutaneous coronary artery damage. In 3 different animal models, we sought to determine whether rapamycin (sirolimus) affects the proliferation and migration of endothelial cells and endothelial progenitor cells. First, after we implanted stents in dogs, we found that re-endothelialization was impeded more by drug-eluting stents than by bare-metal stents, 30 days after percutaneous coronary intervention. Second, in vitro in rats, we found that 1-100 ng/mL of rapamycin time- and dose-dependently inhibited proliferation over 72 hr (with effects evident as early as 24 hr) and also dose-dependently induced endothelial progenitor-cell apoptosis. Finally, in vivo in rats, we observed that vascular endothelial growth factor expression was decreased after 5 days of rapamycin treatment. We conclude that rapamycin impedes re-endothelialization after drug-eluting stent implantation by inhibiting the proliferation and migration of coronary endothelial cells, inducing endothelial progenitor-cell apoptosis, and decreasing vascular endothelial growth factor expression in the circulation.