The largest genome-wide association study for breast cancer in Taiwanese Han population.

PURPOSE: Breast cancer is a molecularly heterogeneous disease, and multiple genetic variants contribute to its development and prognosis. Most of previous genome-wide association studies (GWASs) and polygenic risk scores (PRSs) analyses focused on studying breast cancers of Caucasian populations, which may not be applicable to other population. Therefore, we conducted the largest breast cancer cohort of Taiwanese population to fill in the knowledge gap. METHODS: A total of 152,534 Participants recruited by China Medical University Hospital between 2003 and 2019 were filtered by several patient selection criteria and GWAS quality control steps, resulting in the inclusion of 2496 cases and 9984 controls for this study. We then conducted GWAS for all breast cancers and PRS analyses for all breast cancers and the four breast cancer subtypes, including luminal A, luminal B, basal-like, and HER2-enriched. RESULTS: The GWAS analyses identified 113 SNPs, 50 of which were novel. The PRS models for all breast cancers and the luminal A subtype showed positively correlated trends between the PRS and the risk of developing breast cancer. The odds ratios (95% confidence intervals) for the groups with the highest PRS in all breast cancers and the luminal A subtype were 5.33 (3.79-7.66) and 3.55 (2.13-6.14), respectively. CONCLUSION: In summary, we explored the association of genetic variants with breast cancer in the largest Taiwanese cohort and developed two PRS models that can predict the risk of developing any breast cancer and the luminal A subtype in Taiwanese women.

Age-specific percentile-based prostate-specific antigen cutoff values predict the risk of prostate cancer: A single hospital observation.

Background: Testing for prostate-specific antigen (PSA) is often recommended for men with a potential risk of prostate cancer (PCa) before requiring advanced examination. However, the best PSA cutoff value remains controversial. Object: We compared the predictive performance of age-specific percentile-based PSA thresholds with a conventional cutoff of >4 ng/mL for the risk of PCa. Methods: We included men who received PSA measurements between 2003 and 2017 in a medical center in Taiwan. Logistic regression modeling was used to assess the association between age-specific percentile-based PSA thresholds and PCa risk in age subgroups. We further applied C-statistic and decision curve analysis to compare the predictive performance of age-specific percentile-based PSA with that of a conventional cutoff PSA. Results: We identified 626 patients with PCa and 40 836 patients without PCa. The slope of PSA in patients >60-year-old was almost 3 times that of those <60-year-old (0.713 vs 0.259). The risk effect sizes of the 75th percentile PSA cutoff (<60-year-old: 2.19; 60-70-year-old: 4.36; >70-year-old: 5.84 ng/mL) were comparable to those observed based on the conventional cutoff in all age groups. However, the discrimination performance of the 75th percentile PSA cutoff was better than that of the conventional cutoff among patients aged <60-year-old (C-statistic, 0.783 vs. 0.729, p < 0.05). The 75th percentile cutoffs also correctly identified an additional 2 patients with PCa for every 100 patients with PSA screening at the threshold probability of 20%. Conclusions: Our data support the use of the 75th percentile PSA cutoff to facilitate individualized risk assessment, particularly for patients aged <60-year-old.

Association between coronary artery calcium and all-cause mortality: A large single-center retrospective cohort study.

OBJECTIVE: Previous studies have revealed that coronary artery calcium is related to cardiovascular diseases and mortality. However, most studies have been conducted in Western countries and have excluded patients with pre-existing heart disease. We investigated the association between coronary artery calcium (CAC) and all-cause mortality in an Asian cohort and in subgroups stratified by age, sex, smoking, obesity, diabetes, cardiovascular disease, blood pressure, and biochemical parameters. METHODS: We conducted a retrospective cohort study on 4529 health examinees who underwent multidetector computed tomography in a tertiary medical center in Taiwan between 2011 and 2016. The mean follow-up was 3.5 years. Cox regression was used to estimate the relative hazards of death. Stratified analyses were performed. RESULTS: The all-cause mortality rates were 2.94, 4.88, 17.6, and 33.1 per 1000 person-years for CAC scores of 0, 1-100, 101-400, and >400, respectively. The multivariable adjusted hazard ratios (95% confidence intervals [CIs]) for all-cause mortality were 0.95 (0.53, 1.72), 1.87 (0.89, 3.90), and 3.05 (1.46, 6.39) for CAC scores of 1-100, 101-400, and >400, respectively, relative to a CAC score of 0. Compared with CAC ≤ 400, the HRs (95% CIs) for CAC > 400 were 6.46 (2.44, 17.15) and 1.94 (1.00, 3.76) in younger and older adults, respectively, indicating that age was a moderating variable (p = 0.02). CONCLUSION: High CAC scores were associated with increased all-cause mortality. Although older adult patients had higher risks of death, the relative risk of death for patients with CAC > 400 was more prominent in people younger than 65 years.

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans.

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, ß-galactoside-binding proteins, bind to various gram-negative bacterial LPS, which display ß-galactoside-containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1ß production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis-a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in Drosophila Expressing Mutant Voltage-Gated Sodium Channels.

Voltage-gated sodium (Nav) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Nav-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Nav-channel mutant, paraShu Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses paraShu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Nav-channel mutants, paraGEFS+ and parabss Notably, paraShu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that paraShu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a paraShu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of paraShu phenotypes.

Early Dutasteride Monotherapy in Patients With Elevated Serum Prostate-Specific Antigen Levels Following Robot-Assisted Radical Prostatectomy.

Background: To evaluate the efficacy of early dutasteride administration in patients with a detectable prostate-specific antigen (PSA) levels after robot-assisted radical prostatectomy (RARP). Methods: We retrospectively analyzed RARP patients whose pathological stage is T2a to T3b without lymph node or distant metastasis from 2007 to 2017. All patients received a daily dose of 0.5 mg of dutasteride when post-RARP PSA levels were increasing but had not achieved biochemical recurrence. PSA levels were monitored every 3 months after dutasteride administration. None of the patients received radiotherapy (RT) or androgen-deprivation therapy (ADT) before taking dutasteride. All follow-ups were begun from RARP to January 2019 or to the date of RT/ADT. Results: Thirty-five patients were included in this analysis. The median followed up was 53.6 months. Twenty-two patients (62.9%) showed a PSA response in which the PSA decreased more than 10% at the first follow-up after dutasteride administration. The Pathological stage > T2 (p = 0.012) and positive surgical margin (p = 0.046) were prognostic factors for a PSA response. Twenty-three out of 35 included patients (65.7%) did not require further RT/ADT. The significant risk factor was the PSA level (p = 0.011) at the beginning of dutasteride treatment. The cut-off value of the PSA level to avoid further RT/ADT was 0.195 ng/ml. Conclusions: Early dutasteride administration showed a significant decline in the PSA levels of patients with pathology stage >T2 and positive surgical margin in our retrospective hypothesis-generating study. If dutasteride was provided before the PSA value increased to 0.195 ng/ml after RARP, it would reduce the probability of acquirement of RT/ADT.

Helminth-Induced Production of TGF-ß and Suppression of Graft-versus-Host Disease Is Dependent on IL-4 Production by Host Cells.

Helminths stimulate the secretion of Th2 cytokines, like IL-4, and suppress lethal graft-versus-host disease (GVHD) after bone marrow transplantation. This suppression depends on the production of immune-modulatory TGF-ß and is associated with TGF-ß-dependent in vivo expansion of Foxp3+ regulatory T cells (Treg). In vivo expansion of Tregs is under investigation for its potential as a therapy for GVHD. Nonetheless, the mechanism of induced and TGF-ß-dependent in vivo expansion of Tregs, in a Th2 polarized environment after helminth infection, is unknown. In this study, we show that helminth-induced IL-4 production by host cells is critical to the induction and maintenance of TGF-ß secretion, TGF-ß-dependent expansion of Foxp3+ Tregs, and the suppression of GVHD. In mice with GVHD, the expanding donor Tregs express the Th2-driving transcription factor, GATA3, which is required for helminth-induced production of IL-4 and TGF-ß. In contrast, TGF-ß is not necessary for GATA3 expression by Foxp3+ Tregs or by Foxp3- CD4 T cells. Various cell types of innate or adaptive immune compartments produce high quantities of IL-4 after helminth infection. As a result, IL-4-mediated suppression of GVHD does not require invariant NKT cells of the host, a cell type known to produce IL-4 and suppress GVHD in other models. Thus, TGF-ß generation, in a manner dependent on IL-4 secretion by host cells and GATA3 expression, constitutes a critical effector arm of helminthic immune modulation that promotes the in vivo expansion of Tregs and suppresses GVHD.

Cytosolic galectin-3 and -8 regulate antibacterial autophagy through differential recognition of host glycans on damaged phagosomes.

While glycans are generally displayed on the cell surface or confined within the lumen of organelles, they can become exposed to the cytosolic milieu upon disruption of organelle membrane by various stresses or pathogens. Galectins are a family of ß-galactoside-binding animal lectins synthesized and predominantly localized in the cytosol. Recent research indicates that some galectins may act as "danger signal sensors" by detecting unusual exposure of glycans to the cytosol. Galectin-8 was shown to promote antibacterial autophagy by recognizing host glycans on ruptured vacuolar membranes and interacting with the autophagy adaptor protein NDP52. Galectin-3 also accumulates at damaged phagosomes containing bacteria; however, its functional consequence remains obscure. By studying mouse macrophages infected with Listeria monocytogenes (LM), we showed that endogenous galectin-3 protects intracellular LM by suppressing the autophagic response through a host N-glycan-dependent mechanism. Knock out of the galectin-3 gene resulted in enhanced LC3 recruitment to LM and decreased bacterial replication, a phenotype recapitulated when Galectin-8-deficient macrophages were depleted of N-glycans. Moreover, we explored the concept that alterations in cell surface glycosylation by extracellular factors can be deciphered by cytosolic galectins during the process of phagocytosis/endocytosis, followed by rupture of phagosomal/endosomal membrane. Notably, treatment of cells with sialidase, which removes sialic acid from glycans, resulted in increased galectin-3 accumulation and decreased galectin-8 recruitment at damaged phagosomes, and led to a stronger anti-autophagic response. Our findings demonstrate that cytosolic galectins may sense changes in glycosylation at the cell surface and modulate cellular response through differential recognition of glycans on ruptured phagosomal membranes.

Cumulative Burden of Glucocorticoid-related Adverse Events in Patients with Systemic Lupus Erythematosus: Findings from a 12-year Longitudinal Study.

OBJECTIVE: The aim of this population-based study is to examine the adverse events (AE) associated with longitudinal systemic glucocorticoid (GC) use among an ethnic Chinese systemic lupus erythematosus (SLE) cohort. METHODS: Our study subjects were patients with newly diagnosed SLE aged 18 and older who received at least 1 prescription of systemic GC between 2001 and 2012 from Taiwan's National Health Insurance Research Database (NHIRD). The earliest prescription date of systemic GC for each subject was defined as the index date. For each subject, we calculated the average prednisolone-equivalent dose and the medication possession ratio (MPR) of GC use every 90 days for each patient after the index date. Patients with a diagnosis of AE (defined by the International Classification of Diseases-9-Clinical Modification diagnosis code) during the followup were also identified from the NHIRD. Generalized estimating equations adjusted for propensity score were applied to examine the association between longitudinal GC use and risks of prespecified AE (musculoskeletal, gastrointestinal, ophthalmologic, infectious, cardiovascular, neuropsychiatric, metabolic, and dermatologic diseases). RESULTS: We identified 11,288 patients with SLE (mean followup: 6.28 yrs). Higher doses and higher MPR of GC were associated with increased risk of osteonecrosis [adjusted OR (aOR) 2.87-9.09]. Similar results were found regarding the risk of osteoporosis (aOR 1.71-3.67), bacterial infection (aOR 2.12-3.89), Cushingoid syndrome (aOR 6.51-62.03), and sleep disorder (aOR 1.42-3.59). CONCLUSION: To our knowledge, this is the first study to show that the dose and intensity of longitudinal use of GC were both associated with risk of AE among a nationwide Asian SLE cohort.

Galectin-3 and Its Genetic Variation rs4644 Modulate Enterovirus 71 Infection.

Galectin-3, a chimeric type ß-galactoside-binding protein, is known to modulate viral infection; however, its role in enterovirus 71 (EV71) infection has not been investigated. We generated galectin-3 null rhabdomyosarcoma (RD) cells and evaluated whether EV71 infection would be affected. In galectin-3 null cells, the released and intracellular EV71 viral loads were suppressed after 24 h of infection, and cell death rates were significantly lower, while cell proliferation remained unaltered. In addition, RD cells expressing a nonsynonymous genetic variant of galectin-3, rs4644 (LGALS3 +191C/A, P64H), produced lower virus titers than those with wild-type galectin-3 (C allele). To clarify whether the in vitro viral load reduction correlates with clinical severity, we enrolled children with laboratory-confirmed EV71 infection. Since hyperglycemia is an indicator of severe EV71 infection in children, 152 of 401 enrolled children had glucose examinations at admission, and 59 subjects had serum glucose levels ≥ 150 mg/dL. In comparison to the rs4644 AA genotype (2.2 ± 0.06 log10 mg/dL), serum glucose levels during EV71 infection were higher in patients with CC (2.4 ± 0.17 log10 mg/dL, p = 0.03) and CA (2.4 ± 0.15 log10 mg/dL, p = 0.02) genotypes, respectively. These findings suggest that the rs4644 AA genotype of galectin-3 might exert a protective effect. In summary, galectin-3 affects EV71 replication in our cellular model and its variant, rs4644, is associated with hyperglycemia in the clinical setting. The underlying mechanism and its potential therapeutic application warrant further investigation.

MicroRNA miR-204 and miR-1236 inhibit hepatitis B virus replication via two different mechanisms.

Hepatitis B virus (HBV) is a major human pathogen. In this study, we found that miR-204 and miR-1236 were down-regulated in HBV-producing cells, and each could suppress HBV replication. Using a bioinformatic approach and a reporter assay, we identified miR-1236, which can reduce HBV replication and protein production by directly targeting at HBV specific mRNA. In contrast, miR-204 was identified by a microarray approach, and had no effect on HBV RNA and protein production. Surprisingly, miR-204 could inhibit HBV pregenomic RNA encapsidation and capsid assembly. We further demonstrated that HBV suppressed miR-204 expression via activating a host transcription factor STAT3. We established a positive feed-forward loop between HBV, miR-204 and STAT3. Interestingly, miR-204 has been considered as a tumor suppressor in some literature. Since the risk for hepatocellular carcinoma (HCC) is significantly increased in chronic HBV patients, it is possible that chronic suppression of miR-204 by HBV contributes to HCC incidence. Both miR-204 and miR-1236 might be useful for developing new therapeutics against HBV.

Domperidone, cytochrome P450 3A4 isoenzyme inhibitors and ventricular arrhythmia: a nationwide case-crossover study.

BACKGROUND: Recent evidence suggested that oral form of domperidone may possess pro-arrhythmic effects and increase the risk of ventricular arrhythmia. The concomitant use of cytochrome P450 (CYP) 3A4 isoenzyme inhibitors may further potentiate this association. Nevertheless, empirical data supporting these associations are very limited. The aim of this study was to investigate the association between oral domperidone, CYP 3A4 inhibitors, and ventricular arrhythmia. METHODS: We identified 25,356 patients who were admitted or were seen in the emergency room for ventricular arrhythmia between 2000 and 2011 from Taiwan's Longitudinal Health Insurance Database. We adopted a case-crossover study design to compare the exposure to oral domperidone for the same patient within a "case period" and within a "control period". RESULTS: Conditional logistic regression models showed that domperidone use was significantly associated with an increased odds of ventricular arrhythmia (aOR 1.56, 95%CI [1.41-1.72]). The association was stronger with a higher daily dose of domperidone (>30 mg, aOR 1.98, 95%CI [1.50-2.63]). Furthermore, the co-exposure of domperidone and CYP 3A4 inhibitors was significantly associated with an increased odds of ventricular arrhythmia, especially considering the lasting effect of CYP 3A4 inhibitors (1 day apart: aOR 1.91, 95%CI [1.33-2.75], 3 days apart: aOR 1.90, 95%CI [1.33-2.71], 7 days apart: aOR 1.80, 95%CI [1.28-2.54]). CONCLUSIONS: Our results suggested that oral domperidone was significantly associated with an increased odds of ventricular arrhythmia and that the association was stronger with exposure to >30 mg of domperidone. In addition, our study reported increased odds of ventricular arrhythmia among those who concomitantly used domperidone and CYP 3A4 inhibitors.

Decision to adopt medical technology under the National Health Insurance System in Taiwan: case study of new molecular targeted drugs among non-small cell lung cancer patients.

RATIONALE, AIMS AND OBJECTIVES: Molecular targeted drugs (MTD), gefitinib and erlotinib, have been proven to provide clinical benefits to advanced stage non-small cell lung cancer (NSCLC) patients. Therefore, access to such medical innovations in time is critical for patients with the right indications. The aim of this study was to explore determinants of the adoption of MTD among NSCLC patients under Taiwan's National Health Insurance (NHI) system. METHODS: Using Taiwan's Longitudinal Health Insurance Database and Cancer Registry database as the data source, we identified 1555 newly diagnosed NSCLC patients who started their cancer treatment between 2004 and 2009. Patients were categorized into 'non-MTD' and 'MTD' groups based on the cancer treatment they received. Hierarchical logistic regression was used to explore potential determinants associated with the adoption of MTD by adjusting for patient-, disease-, doctor-, hospital-level characteristics and changes of reimbursement schemes. RESULTS: During the study period, 562 (36%) NSCLC patients were classified as the 'MTD group and 993 (64%) patients were the 'non-MTD' group. Hierarchical logistic regression model showed that the elderly were less likely to receive MTD (OR = 0.41; 95% CI: 0.28-0.60). In contrast, patients whose cancer type was adenocarcinoma (OR = 2.99; 95% CI: 2.09-4.26) were more likely to receive MTD. Those who went to private hospitals (OR = 1.75; 95% CI: 1.01-3.03) and hospitals with higher economic scale (OR = 3.00; 95% CI: 1.72-5.25) were more likely to receive MTD as well. CONCLUSIONS: Our findings suggest that both patient- and hospital-level characteristics significantly affected the adoption of MTD among NSCLC patients.

Intestinal helminths regulate lethal acute graft-versus-host disease and preserve the graft-versus-tumor effect in mice.

Donor T lymphocyte transfer with hematopoietic stem cells suppresses residual tumor growth (graft-versus-tumor [GVT]) in cancer patients undergoing bone marrow transplantation (BMT). However, donor T cell reactivity to host organs causes severe and potentially lethal inflammation called graft-versus-host disease (GVHD). High-dose steroids or other immunosuppressive drugs are used to treat GVHD that have limited ability to control the inflammation while incurring long-term toxicity. Novel strategies are needed to modulate GVHD, preserve GVT, and improve the outcome of BMT. Regulatory T cells (Tregs) control alloantigen-sensitized inflammation of GVHD, sustain GVT, and prevent mortality in BMT. Helminths colonizing the alimentary tract dramatically increase the Treg activity, thereby modulating intestinal or systemic inflammatory responses. These observations led us to hypothesize that helminths can regulate GVHD and maintain GVT in mice. Acute GVHD was induced in helminth (Heligmosomoides polygyrus)-infected or uninfected BALB/c recipients of C57BL/6 donor grafts. Helminth infection suppressed donor T cell inflammatory cytokine generation and reduced GVHD-related mortality, but maintained GVT. H. polygyrus colonization promoted the survival of TGF-ß-generating recipient Tregs after a conditioning regimen with total body irradiation and led to a TGF-ß-dependent in vivo expansion/maturation of donor Tregs after BMT. Helminths did not control GVHD when T cells unresponsive to TGF-ß-mediated immune regulation were used as donor T lymphocytes. These results suggest that helminths suppress acute GVHD using Tregs and TGF-ß-dependent pathways in mice. Helminthic regulation of GVHD and GVT through intestinal immune conditioning may improve the outcome of BMT.

Risk of hospitalization and healthcare cost associated with Diabetes Complication Severity Index in Taiwan's National Health Insurance Research Database.

OBJECTIVE: The aim of this study is to test the validity of adapted Diabetes Complication Severity Index (aDCSI) in predicting the risk of hospitalization and healthcare cost in type 2 diabetic patients using a nationally-representative claims database. STUDY DESIGN: Retrospective cohort study used 4years of claims data from Taiwan's National Health Insurance Research Database (NHIRD). METHODS: Type 2 diabetic patients who had 4-years of enrollment were identified as study subjects (N=136,372). The aDCSI score (sum of diabetic complication with severity levels, range 0-13) and complication count (sum of diabetic complications, range 0-7) were generated using diagnostic codes for each patient. Poisson model and linear regression model were conducted to predict risk of hospitalization and healthcare costs associated with aDCSI score and count of diabetic complications. RESULTS: The aDCSI score (risk ratio 1.51 to 10.32 categorically, and 1.41 linearly) and count of diabetic complications (risk ratio 1.56 to 12.20 categorically, and 1.66 linearly) were significantly positively associated with risk of hospitalization. A one-point increase in the aDCSI score was positively associated with increased healthcare costs. CONCLUSIONS: The performance of aDCSI in predicting risk of hospitalization and healthcare cost in the nationally-representative claims database is similar to those reported in the original study. It may serve as an efficient tool for stratifying type 2 diabetic patients for disease management programs and population-based studies.

Simvastatin downregulates the expression of hepcidin and erythropoietin in HepG2 cells.

Statin therapy may improve responsiveness to erythropoietin-stimulating agents in patients with end-stage renal disease. Although statins increase hepatic iron uptake and storage capacity in cholestatic rats, the underlying mechanisms are unclear. Therefore, we examined the effects of a statin (simvastatin) on the expression of hepcidin, erythropoietin receptor (EPOR) and erythropoietin (EPO) in cultured HepG2 cells. HepG2 cells (6-6.5 × 10(5) cells) were seeded in 6-cm dishes and incubated overnight. The cells were then treated with 0, 0.5, 1, 3, 5, or 10 µM simvastatin, and the mRNA expression of hepcidin, EPOR, and EPO was determined. Data were collected from three independent experiments. The cDNA extracted from the cells was used as a template for real-time polymerase chain reaction, and each sample was tested in duplicate. Significant differences (P < 0.05) among groups were determined using one-way analysis of variance with Fisher's least significant difference post hoc test. Data were adjusted using Bonferroni's method. The relative mRNA expression of hepcidin in HepG2 cells treated with 0.5, 1, 3, 5, and 10 µM simvastatin, relative to the control group, was 0.7273, 0.3303, 0.2418, 0.4131, and 0.4064, respectively. The relative mRNA expression of EPOR was 0.5196, 0.2319, 0.2398, 0.4253, and 0.1245, respectively, while that of EPO was 0.9751, 0.4712, 0.4613, 0.4875, and 0.1654. There was a reverse dose-dependent effect of simvastatin. These results suggest that statins increase erythropoiesis by targeting hepcidin and iron regulatory pathways, independent of erythropoietin.

MicroRNA-22 can reduce parathymosin expression in transdifferentiated hepatocytes.

Pancreatic acinar cells AR42J-B13 can transdifferentiate into hepatocyte-like cells permissive for efficient hepatitis B virus (HBV) replication. Here, we profiled miRNAs differentially expressed in AR42J-B13 cells before and after transdifferentiation to hepatocytes, using chip-based microarray. Significant increase of miRNA expression, including miR-21, miR-22, and miR-122a, was confirmed by stem-loop real-time PCR and Northern blot analyses. In contrast, miR-93, miR-130b, and a number of other miRNAs, were significantly reduced after transdifferentiation. To investigate the potential significance of miR-22 in hepatocytes, we generated cell lines stably expressing miR-22. By 2D-DIGE, LC-MS/MS, and Western blot analyses, we identified several potential target genes of miR-22, including parathymosin. In transdifferentiated hepatocytes, miR-22 can inhibit both mRNA and protein expression of parathymosin, probably through a direct and an indirect mechanism. We tested two computer predicted miR-22 target sites at the 3' UTR of parathymosin, by the 3' UTR reporter gene assay. Treatment with anti-miR-22 resulted in significant elevation of the reporter activity. In addition, we observed an in vivo inverse correlation between miR-22 and parathymosin mRNA in their tissue distribution in a rat model. The phenomenon that miR-22 can reduce parathymosin protein was also observed in human hepatoma cell lines Huh7 and HepG2. So far, we detected no major effect on several transdifferentiation markers when AR42J-B13 cells were transfected with miR-22, or anti-miR-22, or a parathymosin expression vector, with or without dexamethasone treatment. Therefore, miR-22 appears to be neither necessary nor sufficient for transdifferentiation. We discussed the possibility that altered expression of some other microRNAs could induce cell cycle arrest leading to transdifferentiation.

Lateral compartmentalization of T cell receptor versus CD45 by galectin-N-glycan binding and microfilaments coordinate basal and activation signaling.

Lateral compartmentalization of membrane proteins into microdomains regulates signal transduction; however, structural determinants are incompletely understood. Membrane glycoproteins bind galectins in proportion to the number (i.e. NX(S/T) sites) and degree of GlcNAc branching within attached N-glycans, forming a molecular lattice that negatively regulates T cell function and autoimmunity. We find that in resting T cells, partition of CD45 inside and T cell receptor (TCR)/CD4-Lck/Zap-70 outside microdomains is positively and negatively regulated by the galectin lattice and actin cytoskeleton, respectively. In the absence of TCR ligands, the galectin lattice counteracts F-actin to retain CD45 in microdomains while concurrently blocking TCR/CD4-Lck/Zap-70 partition to microdomains by preventing a conformational change in the TCR that recruits Nck/Wiscott Aldrich Syndrome (WASp)/SLP76/F-actin/CD4 to TCR. The counterbalancing activities of the galectin lattice and actin cytoskeleton negatively and positively regulate Lck activity in resting cells and CD45 versus TCR clustering and signaling at the early immune synapse, respectively. Microdomain-localized CD45 inactivates Lck and inhibits TCR signaling at the early immune synapse. Thus, the galectin lattice and actin cytoskeleton interact on opposing sides of the plasma membrane to control microdomain structure and function, coupling basal growth signaling with thresholds to activation.

Peritoneal transport characteristics and dwelling time significantly impact ghrelin clearance in peritoneal dialysis patients.

BACKGROUND: Plasma ghrelin exerts widespread bioactivities. Although it is effectively removed from the blood by a single course of haemodialysis, peritoneal clearance of ghrelin is uncertain. Our study aimed to determine (i) whether there is a correlation between plasma ghrelin levels and characteristics of peritoneal ghrelin clearance, and (ii) whether plasma ghrelin levels significantly impact markers of mortality or morbidity in continuous ambulatory peritoneal dialysis (CAPD) patients. METHODS: We enrolled 50 qualified CAPD patients. Blood was drawn during the fasting state and 2 h post-prandially. Also during these periods, peritoneal effluents were collected for radioimmunoassay of total plasma ghrelin level and measurement of other parameters. Twenty-four hour ascites were collected for determination of ghrelin daily mass transfer. RESULTS: Peritoneal ghrelin clearance was positively correlated with the dialysate-peritoneal creatinine (D/P(Cr)) ratio. Fasting plasma ghrelin levels were inversely correlated with the peritoneal/plasma (D/P(ghrelin)) ratio (P = 0.045). Plasma ghrelin levels were negatively correlated with body mass index, waist-hip ratio, fasting insulin and triglyceride level, and positively correlated with lean body mass. Plasma ghrelin levels were positively correlated with left ventricular mass (LVM), left ventricular mass index and blood pressure. CONCLUSIONS: Peritoneal transporter characteristics may modulate plasma ghrelin levels in CAPD subjects. By contributing to the level of plasma ghrelin, dwelling time may have an impact on LVM and associated morbidity in CAPD patients.

Induction of global stress response in Saccharomyces cerevisiae cells lacking telomerase.

Cellular senescence is a major intermediate step from healthy cells toward tumor cells. By using microarrays that simultaneously examine the transcription levels of 6,200 Saccharomyces cerevisiae genes, we show that 45 gene transcript levels are increased and 11 are decreased after exposure to telomere shortening and cellular senescence in a telomerase-deficient mutant. About half of the genes that showed increased expression were found induced under stress, consistent with the notion that critical short telomeres cause stress to cells. Surprisingly, the expression level of telomere recombination genes was not altered suggesting that even though recombination is a means to rescue critically short telomeres, its machinery was not controlled by telomere shortening. The expression of telomere-proximal genes was also analyzed. The possibility of induction of a program to cope with cellular senescence and active telomere-telomere recombination is discussed.