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1.  Fatty Acid Oxidation Disorders in a Chinese Population in Taiwan 
JIMD Reports  2013;11:165-172.
Background: Fatty acid oxidation (FAO) disorders are a heterogeneous group of inborn errors in the transportation and oxidation of fatty acids. FAO disorders were thought to be very rare in the Chinese population. Newborn screening for FAO disorders beginning in 2002 in Taiwan may have increased the diagnosis of this group of diseases.
Materials and Methods: Till 2012, the National Taiwan University Hospital Newborn Screening Center screened more than 800,000 newborns for FAO disorders. Both patients diagnosed through screening and patients detected after clinical manifestations were included in this study.
Results: A total of 48 patients with FAO disorders were identified during the study period. The disorders included carnitine palmitoyltransferase I deficiency, carnitine acylcarnitine translocase deficiency, carnitine palmitoyltransferase II deficiency, very long-chain acyl-CoA dehydrogenase deficiency, medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenase deficiency, short-chain defects, and carnitine uptake defect. Thirty-nine patients were diagnosed through newborn screening. Five false-negative newborn screening cases were noted during this period, and four patients who were not screened were diagnosed based on clinical manifestations. The ages of all patients ranged from 6 months to 22.9 years (mean age 6.6 years). Except for one case of postmortem diagnosis, there were no other mortalities.
Conclusions: The combined incidence of FAO disorders estimated by newborn screening in the Chinese population in Taiwan is 1 in 20,271 live births. Newborn screening also increases the awareness of FAO disorders and triggers clinical diagnoses of these diseases.
PMCID: PMC3755561  PMID: 23700290
2.  Intermedin in the Paraventricular Nucleus Attenuates Cardiac Sympathetic Afferent Reflex in Chronic Heart Failure Rats 
PLoS ONE  2014;9(4):e94234.
Background and Aim
Intermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide (CGRP) family together with adrenomedullin (AM) and amylin. It has a wide distribution in the central nervous system (CNS) especially in hypothalamic paraventricular nucleus (PVN). Cardiac sympathetic afferent reflex (CSAR) is enhanced in chronic heart failure (CHF) rats. The aim of this study is to determine the effect of IMD in the PVN on CSAR and its related mechanisms in CHF rats.
Methodology/Principal Findings
Rats were subjected to left descending coronary artery ligation to induce CHF or sham-operation (Sham). Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded. CSAR was evaluated by the RSNA and MAP responses to epicardial application of capsaicin. Acute experiments were carried out 8 weeks after coronary ligation or sham surgery under anesthesia. IMD and angiotensin II (Ang II) levels in the PVN were up-regulated in CHF rats. Bilateral PVN microinjection of IMD caused greater decreases in CSAR and the baseline RSNA and MAP in CHF rats than those in Sham rats. The decrease of CSAR caused by IMD was prevented by pretreatment with AM receptor antagonist AM22-52, but not CGRP receptor antagonist CGRP8-37. Ang II in the PVN significantly enhanced CSAR and superoxide anions level, which was inhibited by PVN pretreatment with IMD or tempol (a superoxide anions scavenger) in Sham and CHF rats.
IMD in the PVN inhibits CSAR via AM receptor, and attenuates the effects of Ang II on CSAR and superoxide anions level in CHF rats. PVN superoxide anions involve in the effect of IMD on attenuating Ang II-induced CSAR response.
PMCID: PMC3978024  PMID: 24709972
3.  Functional Connectivity-Based Parcellation of Amygdala Using Self-Organized Mapping: A Data Driven Approach 
Human brain mapping  2013;35(4):1247-1260.
The overall goal of this work is to demonstrate how resting state functional magnetic resonance imaging (fMRI) signals may be used to objectively parcellate functionally heterogeneous subregions of the human amygdala into structures characterized by similar patterns of functional connectivity. We hypothesize that similarity of functional connectivity of subregions with other parts of the brain can be a potential basis to segment and cluster voxels using data driven approaches. In this work, self-organizing map (SOM) was implemented to cluster the connectivity maps associated with each voxel of the human amygdala, thereby defining distinct subregions. The functional separation was optimized by evaluating the overall differences in functional connectivity between the subregions at group level. Analysis of 25 resting state fMRI data sets suggests that SOM can successfully identify functionally independent nuclei based on differences in their inter subregional functional connectivity, evaluated statistically at various confidence levels. Although amygdala contains several nuclei whose distinct roles are implicated in various functions, our objective approach discerns at least two functionally distinct volumes comparable to previous parcellation results obtained using probabilistic tractography and cytoarchitectonic analysis. Association of these nuclei with various known functions and a quantitative evaluation of their differences in overall functional connectivity with lateral orbital frontal cortex and temporal pole confirms the functional diversity of amygdala. The data driven approach adopted here may be used as a powerful indicator of structure–function relationships in the amygdala and other functionally heterogeneous structures as well.
PMCID: PMC3919874  PMID: 23418140
functional connectivity; self-organized mapping; connectivity-based parcellation
4.  Thyroid Endocrine Disruption in Zebrafish Larvae after Exposure to Mono-(2-Ethylhexyl) Phthalate (MEHP) 
PLoS ONE  2014;9(3):e92465.
Phthalates are extensively used as plasticizers in a variety of daily-life products, resulting in widespread distribution in aquatic environments. However, limited information is available on the endocrine disrupting effects of phthalates in aquatic organisms. The aim of the present study was to examine whether exposure to mono-(2-ethylhexyl) phthalate (MEHP), the hydrolytic metabolite of di-(2-ethylhexyl) phthalate (DEHP) disrupts thyroid endocrine system in fish. In this study, zebrafish (Danio rerio) embryos were exposed to different concentrations of MEHP (1.6, 8, 40, and 200 μg/L) from 2 h post-fertilization (hpf) to 168 hpf. The whole-body content of thyroid hormone and transcription of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis were examined. Treatment with MEHP significantly decreased whole-body T4 contents and increased whole-body T3 contents, indicating thyroid endocrine disruption. The upregulation of genes related to thyroid hormone metabolism (Dio2 and UGT1ab) might be responsible for decreased T4 contents. Elevated gene transcription of Dio1 was also observed in this study, which might assist to degrade increased T3 contents. Exposure to MEHP also significantly induced transcription of genes involved in thyroid development (Nkx2.1 and Pax8) and thyroid hormone synthesis (TSHβ, NIS and TG). However, the genes encoding proteins involved in TH transport (transthyretin, TTR) was transcriptionally significantly down-regulated after exposure to MEHP. Overall, these results demonstrate that acute exposure to MEHP alters whole-body contents of thyroid hormones in zebrafish embryos/larvae and changes the transcription of genes involved in the HPT axis, thus exerting thyroid endocrine toxicity.
PMCID: PMC3962405  PMID: 24658602
5.  Development of Rapid Immunochromatographic Test for Hemagglutinin Antigen of H7 Subtype in Patients Infected with Novel Avian Influenza A (H7N9) Virus 
PLoS ONE  2014;9(3):e92306.
Since human infection with the novel H7N9 avian influenza virus was identified in China in March 2013, the relatively high mortality rate and possibility of human-to-human transmission have highlighted the urgent need for sensitive and specific assays for diagnosis of H7N9 infection.
Methodology/Principal Findings
We developed a rapid diagnostic test for the novel avian influenza A (H7N9) virus using anti-hemagglutinin (HA) monoclonal antibodies specifically targeting H7 in an immunochromatographic assay system. The assay limit of detection was 103.5 pfu/ml or 103TCID50 of H7N9 virus. The assay specifically detected H7N9 viral isolates and recombinant HA proteins of H7 subtypes including H7N7 and H7N9, but did not react with non-H7 subtypes including H1N1, H3N2, H5N1, H5N9, and H9N2. The detection sensitivity was 59.4% (19/32) for H7N9 patients confirmed by RT-PCR. Moreover, the highest sensitivity of 61.5% (16/26) was obtained when testing H7N9 positive sputum samples while 35.7% (5/14) of nasopharyngeal swabs and 20% (2/10) of fecal samples tested positive. No false positive detection was found when testing 180 H7N9 negative samples.
Our novel rapid assay can specifically detect H7 HA antigen, facilitating rapid diagnosis for prevention and control of the on-going H7N9 epidemic.
PMCID: PMC3960227  PMID: 24647358
6.  Palmitoyl Acyltransferase, Zdhhc13, Facilitates Bone Mass Acquisition by Regulating Postnatal Epiphyseal Development and Endochondral Ossification: A Mouse Model 
PLoS ONE  2014;9(3):e92194.
ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.
PMCID: PMC3956893  PMID: 24637783
7.  SDF-1/CXCR4 Signaling Preserves Microvascular Integrity and Renal Function in Chronic Kidney Disease 
PLoS ONE  2014;9(3):e92227.
The progressive decline of renal function in chronic kidney disease (CKD) is characterized by both disruption of the microvascular architecture and the accumulation of fibrotic matrix. One angiogenic pathway recently identified as playing an essential role in renal vascular development is the stromal cell-derived factor-1α (SDF-1)/CXCR4 pathway. Because similar developmental processes may be recapitulated in the disease setting, we hypothesized that the SDF-1/CXCR4 system would regulate microvascular health in CKD. Expression of CXCR4 was observed to be increased in the kidneys of subtotally nephrectomized (SNx) rats and in biopsies from patients with secondary focal segmental glomerulosclerosis (FSGS), a rodent model and human correlate both characterized by aberration of the renal microvessels. A reno-protective role for local SDF-1/CXCR4 signaling was indicated by i) CXCR4-dependent glomerular eNOS activation following acute SDF-1 administration; and ii) acceleration of renal function decline, capillary loss and fibrosis in SNx rats treated with chronic CXCR4 blockade. In contrast to the upregulation of CXCR4, SDF-1 transcript levels were decreased in SNx rat kidneys as well as in renal fibroblasts exposed to the pro-fibrotic cytokine transforming growth factor β (TGF-β), the latter effect being attenuated by histone deacetylase inhibition. Increased renal SDF-1 expression was, however, observed following the treatment of SNx rats with the ACE inhibitor, perindopril. Collectively, these observations indicate that local SDF-1/CXCR4 signaling functions to preserve microvascular integrity and prevent renal fibrosis. Augmentation of this pathway, either purposefully or serendipitously with either novel or existing therapies, may attenuate renal decline in CKD.
PMCID: PMC3956917  PMID: 24637920
8.  HIF-1 Alpha Overexpression Correlates with Poor Overall Survival and Disease-Free Survival in Gastric Cancer Patients Post-Gastrectomy 
PLoS ONE  2014;9(3):e90678.
Overall, gastric cancer prognosis remains poor. Detailed characterization of molecular markers that govern gastric cancer pathogenesis is warranted to establish innovative therapeutic options. HIF-1α overexpression has been linked to poor gastric cancer prognosis. However, though researched for years, the prognostic role of HIF-1α in gastric cancer is still controversial. Hence, the objective of the present study was to analyze the prognostic values of HIF-1α, TGF-β, VEGF and pERK1/2 in gastric cancer patients following gastrectomy.
This study included 446 patients with confirmed gastric cancer who underwent gastrectomy in a single Chinese Cancer Center between 2005 and 2006. Clinicopathologic features, as well as immunohistochemical analysis of TGF-β, HIF-1α, VEGF and pERK1/2 were determined. Long-term survival of these patients was analyzed using univariate and multivariate analyses.
HIF-1α overexpression was more frequent in patients with hepatic metastases (71.6% versus 43.0% in those without hepatic metastases, P = 0.000, χ2 = 23.086) and more frequent in patients with peritoneum cavity metastasis (62.3% versus 43.0% in those without such metastasis, P = 0.000, χ2 = 13.691). In univariate analysis, patients with HIF-1α overexpression had a shorter disease-free survival (DFS) and overall survival (OS) than patients with weak-expression (DFS: NA VS. 16.8 m, P = 0.000, χ2 = 74.937; OS: NA VS. 25.5 m, P = 0.000, χ2 = 90.594). Importantly, HIF-1α overexpression was a promising prognostic marker for poor survival by multivariate analysis (DFS: HR 2.766, 95%CI 2.136–2.583, P = 0.000; OS: HR 3.529, 95%CI 2.663–4.667, P = 0.000).
HIF-1α overexpression could be considered a useful independent prognostic biomarker in gastric cancer after gastrectomy, and is correlated to both a poor overall survival and disease-free survival in these patients. HIF-1α expression can be used to stratify patients at higher risk for poor prognosis, and is potentially an important therapeutic target in gastric cancer patients.
PMCID: PMC3948685  PMID: 24614305
9.  Peliosis hepatis complicated by portal hypertension following renal transplantation 
Peliosis hepatis (PH) is a vascular lesion of the liver that mimics a hepatic tumor. PH is often associated with underlying conditions, such as chronic infection and tumor malignancies, or with the use of anabolic steroids, immunosuppressive drugs, and oral contraceptives. Most patients with PH are asymptomatic, but some present with abdominal distension and pain. In some cases, PH may induce intraperitoneal hemorrhage and portal hypertension. This study analyzed a 46-year-old male who received a transplanted kidney nine years prior and had undergone long-term immunosuppressive therapy following the renal transplantation. The patient experienced progressive abdominal distention and pain in the six months prior to this study. Initially, imaging studies revealed multiple liver tumor-like abnormalities, which were determined to be PH by pathological analysis. Because the hepatic lesions were progressively enlarged, the patient suffered from complications related to portal hypertension, such as intense ascites and esophageal varices bleeding. Although the patient was scheduled to undergo liver transplantation, he suffered hepatic failure and died prior to availability of a donor organ.
PMCID: PMC3942847  PMID: 24605041
Peliosis hepatis; Liver neoplasm; Portal hypertension; Renal failure; Renal transplantation
10.  ABCC5, a Gene That Influences the Anterior Chamber Depth, Is Associated with Primary Angle Closure Glaucoma 
PLoS Genetics  2014;10(3):e1004089.
Anterior chamber depth (ACD) is a key anatomical risk factor for primary angle closure glaucoma (PACG). We conducted a genome-wide association study (GWAS) on ACD to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size = −0.045 mm, P = 8.17×10−9). This locus was associated with an increase in risk of PACG in a separate case-control study of 4,276 PACG cases and 18,801 controls (per-allele OR = 1.13 [95% CI: 1.06–1.22], P = 0.00046). The association was strengthened when a sub-group of controls with open angles were included in the analysis (per-allele OR = 1.30, P = 7.45×10−9; 3,458 cases vs. 3,831 controls). Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants influencing anterior chamber dimensions.
Author Summary
The anterior chamber is the space within the eye which is bound by the cornea, and the anterior surfaces of the iris and lens. Anterior chamber depth (ACD) is the distance measured along the eye's optical axis, from the cornea to the lens surface. ACD is an important risk factor for primary angle closure glaucoma (PACG), a major cause of irreversible blindness worldwide, and in particular, individuals of Asian ethnicity. In order to identify the genes that underlie PACG susceptibility, we conducted a two-staged study. We first conducted a large scale genetic study on a total of 5,308 population-based individuals of Asian descent to identify the genetic variants that influence ACD. This was followed by testing for associations between the identified genetic variant and PACG in another independent collection of 4,276 PACG cases and 18,801 controls. We found that a genetic variant within ABCC5 was associated with an increased risk of having PACG. Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants that influence the anterior chamber dimensions of the eye.
PMCID: PMC3945113  PMID: 24603532
11.  Fine-Mapping of Immunodominant Linear B-Cell Epitopes of the Staphylococcus Aureus SEB Antigen Using Short Overlapping Peptides 
PLoS ONE  2014;9(3):e90445.
Staphylococcal enterotoxin B (SEB) is one of the most potent Staphylococcus aureus exotoxins (SEs). Due to its conserved sequence and stable structure, SEB might be a good candidate antigen for MRSA vaccines. Although cellular immune responses to SEB are well-characterized, much less is known regarding SEB-specific humoral immune responses, particularly regarding detailed epitope mapping. In this study, we utilized a recombinant nontoxic mutant of SEB (rSEB) and an AlPO4 adjuvant to immunize BALB/c mice and confirmed that rSEB can induce a high antibody level and effective immune protection against MRSA infection. Next, the antisera of immunized mice were collected, and linear B cell epitopes within SEB were finely mapped using a series of overlapping synthetic peptides. Three immunodominant B cell epitopes of SEB were screened by ELISA, including a novel epitope, SEB205-222, and two known epitopes, SEB97–114 and SEB247-261. Using truncated peptides, an ELISA was performed with peptide-KLH antisera, and the core sequence of the three immunodominant B cell epitopes were verified as SEB97-112, SEB207-222, and SEB247-257. In vitro, all of the immunodominant epitope-specific antisera (anti-SEB97-112, anti-SEB207-222 and anti-SEB247-257) were observed to inhibit SEB-induced T cell mitogenesis and cytokine production from splenic lymphocytes of BALB/c mice. The homology analysis indicated that SEB97–112 and SEB207-222 were well-conserved among different Staphylococcus aureus strains. The 3D crystal structure of SEB indicated that SEB97–112 was in the loop region inside SEB, whereas SEB207-222 and SEB247-257 were in the β-slice region outside SEB. In summary, the fine-mapping of linear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEB immunity against MRSA infection further and will be helpful to optimize MRSA vaccine designs that are based on the SEB antigen.
PMCID: PMC3943954  PMID: 24599257
12.  Epidemiological and Clinical Characteristics and Risk Factors for Death of Patients with Avian Influenza A H7N9 Virus Infection from Jiangsu Province, Eastern China 
PLoS ONE  2014;9(3):e89581.
A novel avian influenza A (H7N9) virus has caused great morbidity as well as mortality since its emergence in Eastern China in February 2013. However, the possible risk factors for death are not yet fully known.
Methods and Findings
Patients with H7N9 virus infection between March 1 and August 14, 2013 in Jiangsu province were enrolled. Data were collected with a standard form. Mean or percentage was used to describe the features, and Fisher's exact test or t-test test was used to compare the differences between fatal and nonfatal cases with H7N9 virus infection. A total of 28 patients with H7N9 virus infection were identified among whom, nine (32.1%) died. The median age of fatal cases was significant higher than nonfatal cases (P<0.05). Patients with older age were more strongly associated with increased odds of death (OR = 30.0; 95% CI, 2.85–315.62). Co-morbidity with chronic lung disease and hypertension were risk factors for mortality (OR = 14.40; 95% CI, 1.30–159.52, OR = 6.67; 95% CI, 1.09–40.43, respectively). Moreover, the presence of either bilateral lung inflammation or pulmonary consolidation on chest imaging on admission was related with fatal outcome (OR = 7.00; 95%CI, 1.10–44.61). Finally, dynamic monitoring showed that lymphopenia was more significant in fatal group than in nonfatal group from day 11 to week five (P<0.05). The decrease in oxygenation indexes were observed in most cases and more significantly in fatal cases after week three (P<0.05), and the value of nearly all fatal cases were below 200 mmHg during our evaluation period.
Among cases with H7N9 virus infection, increased age accompanied by co-morbidities was the risk of death. The severity of lung infection at admission, the persistence of lymphocytopenia, and the extended duration of lower oxygenation index all contributed to worsened outcomes of patients with H7N9 virus infection.
PMCID: PMC3942409  PMID: 24595034
13.  Variations in the MHC Region Confer Risk to Esophageal Squamous Cell Carcinoma on the Subjects from High-Incidence Area in Northern China 
PLoS ONE  2014;9(3):e90438.
The human major histocompatibility complex (MHC) is the most important region in vertebrate genome, and is crucial in innate immunity. Recent studies have demonstrated the possible role of polymorphisms in the MHC region to high risk for esophageal squamous cell carcinoma (ESCC). Our previous genome-wide association study (GWAS) has indicated that the MHC region may confer important risk loci for ESCC, but without further fine mapping. The aim of this study is to further identify the risk loci in the MHC region for ESCC in Chinese population.
Conditional logistic regression analysis (CLRA) was performed on 24 single nucleotide polymorphisms (SNPs) within the MHC region, which were obtained from the genetically matched 937 cases and 692 controls of Chinese Han population. The identified promising SNPs were further correlated with clinical and clinicopathology characteristics. Immunohistochemistry was performed to explore the protein expression pattern of the related genes in ESCC and neighboring normal tissues.
Of the 24 promising SNPs analyzed, we identified three independent SNPs in the MHC region associated with ESCC: rs35399661 (P = 6.07E-06, OR = 1.71, 95%CI = 1.36–2.17), rs3763338 (P = 1.62E-05, OR = 0.63, 95%CI = 0.50–0.78) and rs2844695 (P = 7.60E-05, OR = 0.74, 95%CI = 0.64–0.86). These three SNPs were located at the genes of HLA-DQA1, TRIM27, and DPCR1, respectively. Further analyses showed that rs2844695 was preferentially associated with younger ESCC cases (P = 0.009). The positive immunostaining rates both for HLA-DQA1 and TRIM27 were much higher in ESCC tissues than in neighboring normal tissues (69.4% vs. 26.8% for HLA-DQA1 and 77.6% vs. 47.8% for TRIM27, P<0.001). Furthermore, the overexpression of HLA-DQA1 is correlated significantly with age (P = 0.001) and family history (P<0.001).
This study for the first time provides evidence that multiple genetic factors within the MHC region confer risk to ESCC on the subjects from high-risk area in northern China.
PMCID: PMC3942432  PMID: 24595008
14.  Mst1 and Mst2 Are Essential Regulators of Trophoblast Differentiation and Placenta Morphogenesis 
PLoS ONE  2014;9(3):e90701.
The placenta is essential for survival and growth of the fetus because it promotes the delivery of nutrients and oxygen from the maternal circulation as well as fetal waste disposal. Mst1 and Mst2 (Mst1/2), key components of the mammalian hpo/Mst signaling pathway, encode two highly conserved Ser/Thr kinases and play important roles in the prevention of tumorigenesis and autoimmunity, control of T cell development and trafficking, and embryonic development. However, their functions in placental development are not fully understood, and the underlying cellular and molecular mechanisms remain elusive. Here, we investigated the functions of Mst1/2 in mouse placental development using both conventional and conditional (endothelial) Mst1/2 double knockout mice. We found that the number of trophoblast giant cells dramatically increased while spongiotrophoblast cells almost completely disappeared in Mst1/2 deficient placentas. We showed that Mst1/2 deficiency down regulated the expression of Mash2, which is required for suppressing the differentiation of trophoblast giant cells. Furthermore, we demonstrated that endothelial-specific deletion of Mst1/2 led to impaired placental labyrinthine vasculature and embryonic lethality at E11.5, but neither affected vasculature in yolk sac and embryo proper nor endocardium development. Collectively, our findings suggest that Mst1/2 regulate placental development by control of trophoblast cell differentiation and labyrinthine vasculature at midgestation and Mst1/2 control labyrinth morphogenesis in trophoblast- and fetal endothelial-dependent manners. Thus, our studies have defined novel roles of Mst1/2 in mouse placental development.
PMCID: PMC3942462  PMID: 24595170
15.  Hydrogen Sulfide Inhibits Formaldehyde-Induced Endoplasmic Reticulum Stress in PC12 Cells by Upregulation of SIRT-1 
PLoS ONE  2014;9(2):e89856.
Formaldehyde (FA), a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S), the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER) stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1), a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action.
We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis.
Principal Findings
We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor) significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1.
These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity.
PMCID: PMC3938548  PMID: 24587076
16.  Assessing Interactions of Two Loci (rs4242382 and rs10486567) in Familial Prostate Cancer: Statistical Evaluation of Epistasis 
PLoS ONE  2014;9(2):e89508.
Understanding the impact of multiple genetic variants and their interactions on the disease penetrance of familial multiple prostate cancer is very relevant to the overall understanding of carcinogenesis. We assessed the joint effect of two loci on rs4242382 at 8q24 and rs10486567 at 7p15.2 to this end. We analyzed the data from a Finnish family-based genetic study, which was composed of 947 men including 228 cases in 75 families, to evaluate the respective effects of the two loci on the disease penetrance; in particular, the occurrence and number of prostate cancer cases within a family were utilized to evaluate the interactions between the two loci under the additive and multiplicative Poisson regression models. The risk alleles A at rs4242382 (OR = 1.14, 95% CI 1.08–1.19, P<0.0001) and a risk allele A at rs10486567 (OR = 1.06, 96%CI 1.01–1.11, P = 0.0208) were found to be associated with an increased risk of familial PrCa, especially with four or more cases within a family. A multiplicative model fitted the joint effect better than an additive model (likelihood ratio test X2 = 13.89, P<0.0001). The influence of the risk allele A at rs10486567 was higher in the presence of the risk allele A at rs4242382 (OR = 1.09 (1.01–1.18) vs. 1.01 (0.95–1.07)). Similar findings were observed in non-aggressive PrCa, but not in aggressive PrCa. We demonstrated that two loci (rs4242382 and rs10486567) are highly associated with familial multiple PrCa, and the gene-gene interaction or statistical epistasis was consistent with the Fisher's multiplicative model. These loci's association and epistasis were observed for non-aggressive but not for aggressive tumors. The proposed statistical model can be further developed to accommodate multi-loci interactions to provide further insights into epistasis.
PMCID: PMC3934901  PMID: 24586834
17.  rtM204Q May Serve as a Novel Lamivudine-Resistance-Associated Mutation of Hepatitis B Virus 
PLoS ONE  2014;9(2):e89015.
Background and Aims
Lamivudine (LAM) is still widely used for anti-HBV therapy in China. The study aimed to clarify whether a newly-found rtM204Q mutation from patients was associated with the drug resistance.
HBV complete reverse-transcriptase region was screened by direct sequencing and verified by clonal sequencing. Replication-competent plasmids containing patient-derived 1.1mer mutant or wild-type viral genome were constructed and transfected into HepG2 cells. After cultured with or without serially-diluted antiviral drugs, intracellular HBV replicative intermediates were quantitated for calculating the 50% effective concentration of drug (EC50).
A total of 12,000 serum samples of 9,830 patients with chronic HBV infection were screened. rtM204Q mutation was detected in seven LAM-refractory patients. By contrast, rtM204I/rtM204V mutations were detected in 2,502 patients' samples. The rtM204Q emerged either alone or in concomitance with rtM204I/rtM204V, and all were accompanied with virologic breakthrough in clinical course. Clonal sequencing verified that rtM204Q mutant was predominant in viral quasispecies of these samples. Phenotypic analysis showed that rtM204Q mutant had 89.9% of replication capacity and 76-fold increased LAM EC50 of the concomitant wild-type strain. By contrast, rtM204I mutant in the sample had lower replication capacity and higher LAM resistance (46.3% and 1396-fold increased LAM EC50 of the wild-type strain) compared to rtM204Q mutant. rtM204Q mutant was susceptible to adefovir dipivoxil (ADV) in vitro and ADV/ADV+LAM rescue therapy in clinic.
rtM204Q is suggested to be a novel LAM-resistance-associated mutation. It conferred a moderate resistance with higher competent natural replication capacity compared to rtM204I mutation.
PMCID: PMC3933355  PMID: 24586482
18.  Effects of experimental conditions on the morphologies, structures and growth modes of pulsed laser-deposited CdS nanoneedles 
CdS nanoneedles with different morphologies, structures, and growth modes have been grown on Ni-coated Si(100) surface under different experimental conditions by pulsed laser deposition method. The effects of catalyst layer, substrate temperature, and laser pulse energy on the growth of the CdS nanoneedles were studied in detail. It was confirmed that the formation of the molten catalyst spheres is the key to the nucleation of the CdS nanoneedles by observing the morphologies of the Ni catalyst thin films annealed at different substrate temperatures. Both the substrate temperature and laser pulse energy strongly affected the growth modes of the CdS nanoneedles. The secondary growth of the smaller nanoneedles on the top of the main nanoneedles was found at appropriate conditions. A group of more completed pictures of the growth modes of the CdS nanoneedles were presented.
PMCID: PMC3941934  PMID: 24559455
CdS nanoneedles; Substrate temperature; Laser pulse energy; Growth mode; 61.46.-w; 61.46.Km; 68.37.Lp
19.  Interruption and Non-Adherence to Long-Term Adjuvant Hormone Therapy Is Associated with Adverse Survival Outcome of Breast Cancer Women - An Asian Population-Based Study 
PLoS ONE  2014;9(2):e87027.
This study aimed to evaluate the survival rate of women with breast cancer (BC) comparing persistence versus interruption and adherence versus non-adherence to adjuvant hormonal therapy (HT) in Asian population. Newly-diagnosed BC women from 2003 to 2010 were retrospectively identified from the Taiwan National Health Insurance Research Database. HT prescriptions were extracted to define treatment interruption and medication possession ratio. Their impacts on mortality were estimated by Cox regression with time dependent covariates. Interruption (HR: 1.32; 95% CI: 1.20, 1.46; P<0.0001) and non-adherence (HR: 1.45; 95% CI: 1.32, 1.59; P<0.0001) to adjuvant HT were significantly associated with increased mortality. Interruption to tamoxifen in younger patients and in patients receiving surgery (OP) with adjuvant chemotherapy (CT) was associated with increasing mortality rate when compared with their counterparts. Non-adherence to AIs in both younger and senior age groups and in OP with CT group also resulted in increasing risk. Treatment interruption and non-adherence to adjuvant HT were found to be associated with the increasing all-cause mortality of the Asian BC women; a greater impact of interruption and non-adherence on mortality was especially found in the younger BC population.
PMCID: PMC3931619  PMID: 24586261
20.  Intracellular Acid-Extruding Regulators and the Effect of Lipopolysaccharide in Cultured Human Renal Artery Smooth Muscle Cells 
PLoS ONE  2014;9(2):e90273.
Homeostasis of the intracellular pH (pHi) in mammalian cells plays a pivotal role in maintaining cell function. Thus far, the housekeeping Na+-H+ exchanger (NHE) and the Na+-HCO3− co-transporter (NBC) have been confirmed in many mammalian cells as major acid extruders. However, the role of acid-extruding regulators in human renal artery smooth muscle cells (HRASMCs) remains unclear. It has been demonstrated that lipopolysaccharide (LPS)-induced vascular occlusion is associated with the apoptosis, activating calpain and increased [Ca2+]i that are related to NHE1 activity in endothelia cells. This study determines the acid-extruding mechanisms and the effect of LPS on the resting pHi and active acid extruders in cultured HRASMCs. The mechanism of pHi recovery from intracellular acidosis (induced by NH4Cl-prepulse) is determined using BCECF-fluorescence in cultured HRASMCs. It is seen that (a) the resting pHi is 7.19±0.03 and 7.10±0.02 for HEPES- and CO2/HCO3−- buffered solution, respectively; (b) apart from the housekeeping NHE1, another Na+-coupled HCO3− transporter i.e. NBC, functionally co-exists to achieve acid-equivalent extrusion; (c) three different isoforms of NBC: NBCn1 (SLC4A7; electroneutral), NBCe1 (SLC4A4; electrogenic) and NBCe2 (SLC4A5), are detected in protein/mRNA level; and (d) pHi and NHE protein expression/activity are significantly increased by LPS, in both a dose- and time- dependent manner, but NBCs protein expression is not. In conclusion, it is demonstrated, for the first time, that four pHi acid-extruding regulators: NHE1, NBCn1, NBCe1 and NBCe2, co-exist in cultured HRASMCs. LPS also increases cellular growth, pHi and NHE in a dose- and time-dependent manner.
PMCID: PMC3931831  PMID: 24587308
21.  Contamination of Medical Charts: An Important Source of Potential Infection in Hospitals 
PLoS ONE  2014;9(2):e78512.
This prospective study aims to identify and compare the incidence of bacterial contamination of hospital charts and the distribution of species responsible for chart contamination in different units of a tertiary hospital.
All beds in medical, surgical, pediatric, and obstetric-gynecologic general wards (556) and those in corresponding special units (125) including medical, surgical, pediatric intensive care units (ICUs), the obstetric tocolytic unit and delivery room were surveyed for possible chart contamination. The outer surfaces of included charts were sampled by one experienced investigator with sterile cotton swabs rinsed with normal saline.
For general wards and special units, the overall sampling rates were 81.8% (455/556) and 85.6% (107/125) (p = 0.316); the incidence of chart contamination was 63.5% and 83.2%, respectively (p<0.001). Except for obstetric-gynecologic charts, the incidence was significantly higher in each and in all ICUs than in corresponding wards. Coagulase-negative staphylococci was the most common contaminant in general wards (40.0%) and special units (34.6%) (p>0.05). Special units had a significantly higher incidence of bacterial contamination due to Staphylococcus aureus (17.8%), Methicillin-resistant Staphylococcus aureus (9.3%), Streptococcus viridans (9.4%), Escherichia coli (11.2%), Klebsiella pneumoniae (7.5%), and Acinetobacter baumannii (7.5%). Logistic regression analysis revealed the incidence of chart contamination was 2- to 4-fold higher in special units than in general wards [odds ratios: 1.97–4.00].
Noting that most hospital charts are contaminated, our study confirms that a hospital chart is not only a medical record but also an important source of potential infection. The plastic cover of the medical chart can harbor potential pathogens, thus acting as a vector of bacteria. Additionally, chart contamination is more common in ICUs. These findings highlight the importance of effective hand-washing before and after handling medical charts. However, managers and clinical staff should pay more attention to the issue and may consider some interventions.
PMCID: PMC3928153  PMID: 24558355
22.  The epidemiology of gastrointestinal stromal tumors in Taiwan, 1998–2008: a nation-wide cancer registry-based study 
BMC Cancer  2014;14:102.
To investigate the incidence of gastrointestinal stromal tumors (GISTs) in Taiwan and the impact of imatinib on the overall survival (OS) of GIST patients.
GISTs were identified from the Taiwan Cancer Registry (TCR) from 1998 to 2008. The age-adjusted incidence rates and the observed OS rates were calculated. Cox proportional hazards models were applied to examine the mortality risk in three time periods (1998–2001, 2002–2004, 2005–2008) according to the application and availability of imatinib.
From 1998 to 2008, 2,986 GISTs were diagnosed in Taiwan. The incidence increased from 1.13 per 100,000 in 1998 to 1.97 per 100,000 in 2008. The most common sites were stomach (47-59%), small intestine (31-38%), and colon/rectum (6-9%). The 5-year observed OS was 66.5% (60.3% for men, 74.2% for women, P < .0001). GISTs in the stomach had a better 5-year observed OS (69.4%) than those in the small intestine (65.1%) (P < .0001). The outcome of GIST improved significantly after the more widespread use of imatinib; the 5-year observed OS increased from 58.9% during 1998–2001 to 70.2% during 2005–2008 (P < .0001). Younger age, female sex, stomach location, and later diagnostic years were independent predictors of a better survival.
The incidence of GIST has been increasing in Taiwan, partially due to the advancement of diagnostic technology/method and the increased awareness by physicians. The outcome of GIST has improved significantly with the availability and the wider use of imatinib.
PMCID: PMC3932802  PMID: 24548660
Gastrointestinal stromal tumors; Incidence; Imatinib; Survival
23.  Honokiol Protected against Heatstroke-Induced Oxidative Stress and Inflammation in Diabetic Rats 
We aimed at investigating the effect of honokiol on heatstroke in an experimental rat model. Sprogue-Dawley rats were divided into 3 groups: normothermic diabetic rats treated with vehicle solution (NTDR+V), heatstroke-diabetic rats treated with vehicle (HSDR+V), and heatstroke rats treated with konokiol (0.5–5 mg/ml/kg) (HSDR+H). Sixty minutes before the start of heat stress, honokiol or vehicle solution was administered. (HSDR+H) significantly (a) attenuated hyperthermia, hypotension and hypothalamic ischemia, hypoxia, and neuronal apoptosis; (b) reduced the plasma index of the toxic oxidizing radicals; (c) diminished the indices of hepatic and renal dysfunction; (d) attenuated the plasma systemic inflammatory response molecules; (e) promoted plasma levels of an anti-inflammatory cytokine; (f) reduced the index of infiltration of polymorphonuclear neutrophils in the serum; and (g) promoted the survival time fourfold compared with the (HSDR+V) group. In conclusion, honokiol protected against the outcome of heatstroke by reducing inflammation and oxidative stress-mediated multiple organ dysfunction in diabetic rats.
PMCID: PMC3947704
24.  Rosiglitazone Increases Cerebral Klotho Expression to Reverse Baroreflex in Type 1-Like Diabetic Rats 
BioMed Research International  2014;2014:309151.
Reduced baroreflex sensitivity (BRS) is widely observed in diabetic human and animals. Rosiglitazone is one of the clinically used thiazolidinediones (TZD) known as PPARγ agonist. Additionally, the klotho protein produced from choroid plexus in the central nervous system is regulated by PPARγ. In an attempt to develop the new therapeutic strategy, we treated streptozotocin-induced diabetic rats (STZ) with rosiglitazone (STZ + TZD) orally at 10 mg/kg for 7 days. Also, STZ rats were subjected to intracerebroventricular (ICV) infusion of recombinant klotho at a dose of 3 μg/2.5 μL via syringe pump (8 μg/hr) daily for 7 days. The BRS and heart rate variability were then estimated under challenge with a depressor dose of sodium nitroprusside (50 μg/kg) or a pressor dose of phenylephrine (8 μg/kg) through an intravenous injection. Lower expression of klotho in medulla oblongata of diabetic rats was identified. Cerebral infusion of recombinant klotho or oral administration of rosiglitazone reversed BRS in diabetic rats. In conclusion, recovery of the decreased klotho in brain induced by rosiglitazone may restore the impaired BRS in diabetic rats. Thus, rosiglitazone is useful to reverse the reduced BRS through increasing cerebral klotho in diabetic disorders.
PMCID: PMC3943406
25.  SPARC silencing inhibits the growth of acute myeloid leukemia transformed from myelodysplastic syndrome via induction of cell cycle arrest and apoptosis 
Secreted protein acidic and rich in cysteine (SPARC) plays key roles in erythropoiesis; haploinsufficiency of SPARC is implicated in the progression of the 5q- syndrome. However, the role of SPARC in other subtypes of myelodysplastic syndrome (MDS) is not fully understood, particularly in the del(5q) type with a complex karyotype, which has a high risk to transform into acute myeloid leukemia (AML). In the present study, we investigated the role of SPARC in the proliferation and apoptosis of SKM-1 cells, an acute myeloid leukemia cell line transformed from an MDS cell line. SKM-1 cells were infected with SPARC-RNAi-LV or NC-GFP-LV lentivirus. Apoptosis and cell cycle profiling were assessed by flow cytometry, and cell proliferation was evaluated by MTS assay. The mRNA and protein expression levels of SPARC, p53, caspase-3, caspase-9 and Fas were detected by RT-PCR, real-time PCR and western blot assay. The SPARC shRNA constructed by us led to a significant reduction in SPARC expression in SKM-1 cells. SPARC knockdown inhibited the proliferation of SKM-1 cells by inducing cell cycle arrest at the G1/G0 phase and apoptosis. SPARC knockdown elevated the expression of p53, caspase-9, caspase-3 and Fas at both the mRNA and protein levels. SPARC silencing inhibited the growth of AML transformed from MDS by activating p53-induced apoptosis and cell cycle arrest. These data indicate that SPARC acts as an oncogene in transformed MDS/AML and is a potential therapeutic target in MDS/AML.
PMCID: PMC3976133  PMID: 24535175
SPARC gene; myelodysplastic syndrome; transfection; the 5q- syndrome

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