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1.  Dietary Restrictions in Dialysis Patients: Is There Anything Left to Eat? 
Seminars in dialysis  2015;28(2):159-168.
A significant number of dietary restrictions are imposed traditionally and uniformly on maintenance dialysis patients, whereas there is very little data to support their benefits. Recent studies indicate that dietary restrictions of phosphorus may lead to worse survival and poorer nutritional status. Restricting dietary potassium may deprive dialysis patients of heart-healthy diets and lead to intake of more atherogenic diets. There is little data about the survival benefits of dietary sodium restriction, and limiting fluid intake may inherently lead to lower protein and calorie consumption, when in fact dialysis patients often need higher protein intake to prevent and correct protein-energy wasting. Restricting dietary carbohydrates in diabetic dialysis patients may not be beneficial in those with burnt-out diabetes. Dietary fat including omega-3 fatty acids may be important caloric sources and should not be restricted. Data to justify other dietary restrictions related to calcium, vitamins and trace elements are scarce and often contradictory. The restriction of eating during hemodialysis treatment is likely another incorrect practice that may worsen hemodialysis induced hypoglycemia and nutritional derangements. We suggest careful relaxation of most dietary restrictions and adoption of a more balanced and individualized approach, thereby easing some of these overzealous restrictions that have not been proven to offer major advantages to patients and their outcomes and which may in fact worsen patients’ quality of life and satisfaction. This manuscript critically reviews the current paradigms and practices of recommended dietary regimens in dialysis patients including those related to dietary protein, carbohydrate, fat, phosphorus, potassium, sodium, and calcium, and discusses the feasibility and implications of adherence to ardent dietary restrictions.
PMCID: PMC4385746  PMID: 25649719
Dietary restriction; dietary protein intake; dietary load; nutritional management; outcomes
2.  Pluripotent Human embryonic stem cell derived neural lineages for in vitro modelling of enterovirus 71 infection and therapy 
Virology Journal  2016;13:5.
The incidence of neurological complications and fatalities associated with Hand, Foot & Mouth disease has increased over recent years, due to emergence of newly-evolved strains of Enterovirus 71 (EV71). In the search for new antiviral therapeutics against EV71, accurate and sensitive in vitro cellular models for preliminary studies of EV71 pathogenesis is an essential prerequisite, before progressing to expensive and time-consuming live animal studies and clinical trials.
This study thus investigated whether neural lineages derived from pluripotent human embryonic stem cells (hESC) can fulfil this purpose. EV71 infection of hESC-derived neural stem cells (NSC) and mature neurons (MN) was carried out in vitro, in comparison with RD and SH-SY5Y cell lines.
Upon assessment of post-infection survivability and EV71 production by the various types, it was observed that NSC were significantly more susceptible to EV71 infection compared to MN, RD (rhabdomyosarcoma) and SH-SY5Y cells, which was consistent with previous studies on mice. The SP81 peptide had significantly greater inhibitory effect on EV71 production by NSC and MN compared to the cancer-derived RD and SH-SY5Y cell lines.
Hence, this study demonstrates that hESC-derived neural lineages can be utilized as in vitro models for studying EV71 pathogenesis and for screening of antiviral therapeutics.
PMCID: PMC4704260  PMID: 26738773
EV71; HFMD; Infectious diseases; Neurons; Viral
3.  Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons 
Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP–GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP–GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP–GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin–GPR54 signaling, which could be a potential pathway to suppress cell migration.
PMCID: PMC4771921  PMID: 26973595
metastasis; cancer; cytoskeleton; preoptic area; reproduction; GPR54
4.  Cycling hypoxia induces chemoresistance through the activation of reactive oxygen species-mediated B-cell lymphoma extra-long pathway in glioblastoma multiforme 
Cycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It contributes to the resistance to cytotoxic therapies through anti-apoptotic effects. However, the mechanism underlying cycling hypoxia-mediated anti-apoptosis remains unclear.
Reactive oxygen species (ROS) production, activation of the hypoxia-inducible factor-1 alpha (HIF-1α) and nuclear factor-κB (NF-κB) signaling pathways, B-cell lymphoma extra-long (Bcl-xL) expression, caspase activation, and apoptosis in in vitro hypoxic stress-treated glioblastoma cells or tumor hypoxic cells derived from human glioblastoma xenografts were determined by in vitro ROS analysis, reporter assay, western blotting analysis, quantitative real-time PCR, caspase-3 activity assay, and annexin V staining assay, respectively. Tempol, a membrane-permeable radical scavenger, Bcl-xL knockdown, and specific inhibitors of HIF-1α and NF-κB were utilized to explore the mechanisms of cycling hypoxia-mediated resistance to temozolomide (TMZ) in vitro and in vivo and to identify potential therapeutic targets.
Bcl-xL expression and anti-apoptotic effects were upregulated under cycling hypoxia in glioblastoma cells concomitantly with decreased responses to TMZ through ROS-mediated HIF-1α and NF-κB activation. Tempol, YC-1 (HIF-1 inhibitor), and Bay 11-7082 (NF-κB inhibitor) suppressed the cycling hypoxia-mediated Bcl-xL induction in vitro and in vivo. Bcl-xL knockdown and Tempol treatment inhibited cycling hypoxia-induced chemoresistance. Moreover, Tempol treatment of intracerebral glioblastoma-bearing mice combined with TMZ chemotherapy synergistically suppressed tumor growth and increased survival rate.
Cycling hypoxia-induced Bcl-xL expression via ROS-mediated HIF-1α and NF-κB activation plays an important role in the tumor microenvironment-promoted anti-apoptosis and chemoresistance in glioblastoma. Thus, ROS blockage may be an attractive therapeutic strategy for tumor microenvironment-induced chemoresistance.
PMCID: PMC4693410  PMID: 26711814
B-cell lymphoma extra-long; Cycling hypoxia; Glioblastoma; Nuclear factor-κb; Reactive oxygen species
5.  Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations 
PLoS ONE  2015;10(12):e0145698.
The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔCq method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.
PMCID: PMC4689371  PMID: 26701781
6.  Association between Thiopurine S-Methyltransferase Polymorphisms and Azathioprine-Induced Adverse Drug Reactions in Patients with Autoimmune Diseases: A Meta-Analysis 
PLoS ONE  2015;10(12):e0144234.
Azathioprine (AZA) is widely used as an immunosuppressive drug in autoimmune diseases, but its use is limited by significant adverse drug reactions (ADRs). Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in AZA metabolism. Several clinical guidelines recommend determining TPMT genotype or phenotype before initiating AZA therapy. Although several studies have investigated the association between TPMT polymorphisms and AZA-induced ADRs, the results are inconsistent. The purpose of this study is to evaluate whether there is an association between TPMT polymorphisms and AZA-induced ADRs using meta-analysis.
We explored PubMed, Web of Science and Embase for articles on TPMT polymorphisms and AZA-induced ADRs. Studies that compared TPMT polymorphisms with-ADRs and without-ADRs in patients with autoimmune diseases were included. Relevant outcome data from all the included articles were extracted and the pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated using Revman 5.3 software.
Eleven published studies, with a total of 651 patients with autoimmune diseases, investigated associations between TPMT polymorphisms and AZA-induced ADRs, were included in this meta-analysis. Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with AZA-induced overall ADRs, bone marrow toxicity and gastric intolerance; pooled ORs were 3.12 (1.48–6.56), 3.76 (1.97–7.17) and 6.43 (2.04–20.25), respectively. TPMT polymorphisms were not associated with the development of hepatotoxicity; the corresponding pooled OR was 2.86 (95%CI: 0.32–25.86). However, the association in GI subset could be driven by one single study. After this study was excluded, the OR was 2.11 (95%CI: 0.36–12.42); namely, the association became negative.
Our meta-analysis demonstrated an association of TPMT polymorphisms with overall AZA-induced ADRs, bone marrow toxicity and gastric intolerance, but not with hepatotoxicity. The presence of the normal TPMT genotypes cannot preclude the development of ADRs during AZA treatment, TPMT genotyping prior to commencing AZA therapy cannot replace, may augment, the current practice of regular monitoring of the white blood cell. Because of small sample sizes, large and extensive exploration was required to validate our findings.
PMCID: PMC4669175  PMID: 26633017
7.  Comparative Mortality-Predictability Using Alkaline Phosphatase and Parathyroid Hormone in Patients on Peritoneal Dialysis and Hemodialysis 
♦ Background: In hemodialysis (HD) patients, serum alkaline phosphatase (ALP) and parathyroid hormone (PTH) derangements are associated with mortality, but outcome-predictability using ALP and PTH in peritoneal dialysis (PD) patients remains uncertain.
♦ Methods: In a cohort of 9244 adult PD patients from a large national dialysis organization (entry period 2001 - 2006, with follow-up through 2009), we used multivariable Cox models adjusted for case-mix and laboratory covariates to examine the associations of time-averaged ALP and PTH with all-cause mortality. We then compared mortality-predictability using ALP and PTH in 9244 PD and 99 323 HD patients.
♦ Results: In PD patients, ALP concentrations exceeding 150 U/L were associated with increased mortality (reference ALP: 70 to <90 U/L). Hazard ratios (HRs) and 95% confidence intervals (CIs) were 1.18 (1.03 to 1.36), 1.27 (1.08 to 1.50), 1.49 (1.23 to 1.79), and 1.35 (1.19 to 1.53) for ALP concentrations of 150 to <170 U/L, 170 to <190 U/L, 190 to <210 U/L, and ≥210 U/L respectively. In contrast, we observed a U-shaped association between PTH concentration and death risk in PD patients, with PTH concentrations of less than 200 pg/mL and 700 pg/mL or more associated with increased mortality (reference PTH: 200 to <300 pg/mL). Hazard ratios and 95% CIs were 1.25 (1.12 to 1.41), 1.12 (1.02 to 1.23), 1.06 (0.96 to 1.18), 1.09 (0.97 to 1.24), 1.12 (0.97 to 1.29), 1.18 (0.99 to 1.40), and 1.23 (1.09 to 1.38) for PTH concentrations of <100 pg/mL, 100 to <200 pg/mL, 300 to <400 pg/mL, 400 to <500 pg/mL, 500 to <600 pg/mL, 600 to <700 pg/mL, and ≥700 pg/mL respectively. Compared with PD patients having serum concentrations of ALP and PTH within reference ranges, patients on HD experienced increased mortality across all ALP and PTH concentrations, particularly those in the lowest and highest categories.
♦ Conclusions: In summary, higher ALP concentrations are associated with increased mortality, and lower and higher PTH concentrations are both associated with death risk in PD patients. The utility of ALP in the management of chronic kidney disease mineral bone disorders in PD patients warrants further study.
PMCID: PMC4269499  PMID: 24385335
CKD mineral bone disorders; alkaline phosphatase; parathyroid hormone
8.  Effects of Adherence to Statin Therapy on Health Care Outcomes and Utilizations in Taiwan: A Population-Based Study 
BioMed Research International  2015;2015:149573.
Aim. Good medication adherence may decrease the probability of worse outcomes and reduce unnecessary medical care costs. This study aims to evaluate medication adherence for people on statin therapy. Methods. National health insurance databases were analyzed from January 1, 2001, to December 31, 2007. Study samples were patients of 45 years and older adults who took statin for the first time during the study period. Medication possession ratio (MPR) was measured until the patients had hospitalization or reached the three-year follow-up period. We identified a good (MPR ≥ 80%) and a poor (MPR < 80%) medication adherence group to conduct statistical analyses. Results. 40.8% of patients were of good medication adherence and 59.2% were of poor medication adherence. Multivariate logistic regression model indicated that the MPR ≥ 80% group had significantly less probability of hospitalization (P < 0.001). Being men, increasing age, higher Charlson Comorbidity Index (CCI) scores, seeking care mostly in the medical center or teaching hospitals, and living in the suburban or rural areas had higher probability of hospitalization (P < 0.05 or P < 0.001). The MPR ≥ 80% group spent less hospitalization expenditures (P < 0.001). Conclusion. Effective interventions may be applied to the poor medication adherence group in order to improve their health care outcomes.
PMCID: PMC4619755  PMID: 26539464
9.  Epi-reevesioside F inhibits Na+/K+-ATPase, causing cytosolic acidification, Bak activation and apoptosis in glioblastoma 
Oncotarget  2015;6(27):24032-24046.
Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.
PMCID: PMC4695168  PMID: 26125228
Epi-reevesioside F; intracellular Na+ concentration; cytosolic acidification; mitochondrial dysfunction; bak activation
10.  Intranasal Delivery of Apelin-13 Is Neuroprotective and Promotes Angiogenesis After Ischemic Stroke in Mice 
ASN NEURO  2015;7(5):1759091415605114.
Apelin is a peptide originally isolated from bovine stomach tissue extracts and identified as an endogenous ligand of the APJ receptor; recent work showed that apelin ameliorates the ischemic injury in the heart and the brain. Being an analogue to the angiotensin II receptor, the apelin/APJ signaling may mediate angiogenesis process. We explored the noninvasive intranasal brain delivery method and investigated therapeutic effects of apelin-13 in a focal ischemic stroke model of mice. Intranasal administration of apelin-13 (4 mg/kg) was given 30 min after the onset of stroke and repeated once daily. Three days after stroke, mice received apelin-13 had significantly reduced infarct volume and less neuronal death in the penumbra. Western blot analyses showed upregulated levels of apelin, apelin receptor APLNR, and Bcl-2 and decreased caspase-3 activation in the apelin-13-treated brain. The proinflammatory cytokines tumor necrosis factor-alpha, interleukin-1β, and chemokine monocyte chemoattractant protein-1 mRNA increased in the ischemic brain, which were significantly attenuated by apelin-13. Apelin-13 remarkably reduced microglia recruitment and activation in the penumbra according to morphological features of Iba-1-positive cells 3 days after ischemia. Apelin-13 significantly increased the expression of angiogenic factor vascular endothelial growth factor and matrix metalloproteinase-9 14 days after stroke. Angiogenesis illustrated by collagen IV + /5-bromo-2′-deoxyuridin + colabeled cells was significantly increased by the apelin-13 treatment 21 days after stroke. Finally, apelin-13 promoted the local cerebral blood flow restoration and long-term functional recovery. This study demonstrates a noninvasive intranasal delivery of apelin-13 after stroke, suggesting that the reduced inflammatory activities, decreased cell death, and increased angiogenesis contribute to the therapeutic benefits of apelin-13.
PMCID: PMC4580122  PMID: 26391329
apelin-13; neuroprotection; angiogenesis; inflammation; functional recovery; ischemic stroke
11.  Synthesis and biological evaluation of panitumumab-IRDye800 conjugate as a fluorescence imaging probe for EGFR-expressing cancers 
MedChemComm  2014;5(9):1337-1346.
To investigate panitumumab-IRDye800 as an intraoperative optical imaging agent for epidermal growth factor receptor (EGFR)-expressing cancers, we developed clinical-quality panitumumab-IRDye800 and evaluated its specificity and sensitivity to visualize tumors by fluorescence imaging in a variety of mouse xenograft models with different levels of EGFR-expression. Panitumumab was chemically conjugated to NIR-dye (Li-COR 800CW) at well-defined and limited substitution ratio (1:1–2) for the characterization of fluorescence signals. Yield and purity of the conjugate was 80±5% and 95±2% respectively (n= 6). Quality control (QC) tests showed that product was suitable for clinical development. Female athymic nude xenograft tumor bearing mice (n=5 per tumor model) with very low (BT-474), moderate (MDA-MB-231), and high (MDA-MB-468) EGFR-expression levels were administered panitumumab-IRDye800 formulations (100 μg of mAb in 100 μL of 0.9% saline) via tail-vein injection. Animal imaging and biodistribution experiments were conducted on the FMT 2500 (Perkin Elmer) fluorescence scanner at 24, 48, 72, 96, and 144 hours post injection. Immuno-fluorescence images of panitumumab-IRDye conjugate recorded in mouse xenograft models showed a good correlation (R2 = 0.91) between EGFR-expression level and tumor uptake. Uptake of panitumumab labeled with IR-Dye or [89Zr] in different tumor xenografts with high, medium, and low EGFR expression, as measured by fluorescence or radioactive counts are highly correlated (r2= 0.99). This preclinical in-vivo study proved that panitumumab-IRDye800 is specific and optical imaging in conjunction with this probe is sensitive enough to detect EGFR-expressing tumors.
PMCID: PMC4241857  PMID: 25431648
12.  Over-Expressed Twist Associates with Markers of Epithelial Mesenchymal Transition and Predicts Poor Prognosis in Breast Cancers via ERK and Akt Activation 
PLoS ONE  2015;10(8):e0135851.
Overexpression of Twist, a highly conserved basic helix-loop-helix transcription factor, is associated with epithelial-mesenchymal transition (EMT) and predicts poor prognosis in various kinds of cancers, including breast cancer. In order to further clarify Twist’s role in breast cancer, we detected Twist expression in breast cancer tissues by immunohistochemistry. Twist expression was observed in 54% (220/408) of breast cancer patients and was positively associated with tumor size, Ki67, VEGF-C and HER2 expression. Conversely, Twist was negatively associated with estrogen receptor (ER), progesterone receptor (PgR) and E-cadherin expression. Patients with Twist expression had a poorer prognosis for 30-month disease free survival (DFS) (82.9%) than patients with negative Twist (92.3%). Overexpression of Twist led to dramatic changes in cellular morphology, proliferation, migratory/invasive capability, and expression of EMT-related biomarkers in breast cancer cells. Moreover, we show that Twist serves as a driver of tumorigenesis, as well as an inducer of EMT, at least in part, through activation of the Akt and extracellular signal-regulated protein kinase (ERK) pathways which are critical for Twist-mediated EMT. Our results demonstrate that Twist expression is an important prognostic factor in breast cancer patients.
PMCID: PMC4546631  PMID: 26295469
13.  Therapeutic Effects of Pharmacologically Induced Hypothermia against Traumatic Brain Injury in Mice 
Journal of Neurotrauma  2014;31(16):1417-1430.
Preclinical and clinical studies have shown therapeutic potential of mild-to-moderate hypothermia for treatments of stroke and traumatic brain injury (TBI). Physical cooling in humans, however, is usually slow, cumbersome, and necessitates sedation that prevents early application in clinical settings and causes several side effects. Our recent study showed that pharmacologically induced hypothermia (PIH) using a novel neurotensin receptor 1 (NTR1) agonist, HPI-201 (also known as ABS-201), is efficient and effective in inducing therapeutic hypothermia and protecting the brain from ischemic and hemorrhagic stroke in mice. The present investigation tested another second-generation NTR1 agonist, HPI-363, for its hypothermic and protective effect against TBI. Adult male mice were subjected to controlled cortical impact (CCI) (velocity=3 m/sec, depth=1.0 mm, contact time=150 msec) to the exposed cortex. Intraperitoneal administration of HPI-363 (0.3 mg/kg) reduced body temperature by 3–5°C within 30–60 min without triggering a shivering defensive reaction. An additional two injections sustained the hypothermic effect in conscious mice for up to 6 h. This PIH treatment was initiated 15, 60, or 120 min after the onset of TBI, and significantly reduced the contusion volume measured 3 days after TBI. HPI-363 attenuated caspase-3 activation, Bax expression, and TUNEL-positive cells in the pericontusion region. In blood–brain barrier assessments, HPI-363 ameliorated extravasation of Evans blue dye and immunoglobulin G, attenuated the MMP-9 expression, and decreased the number of microglia cells in the post-TBI brain. HPI-363 decreased the mRNA expression of tumor necrosis factor-α and interleukin-1β (IL-1β), but increased IL-6 and IL-10 levels. Compared with TBI control mice, HPI-363 treatments improved sensorimotor functional recovery after TBI. These findings suggest that the second generation NTR-1 agonists, such as HPI-363, are efficient hypothermic-inducing compounds that have a strong potential in the management of TBI.
PMCID: PMC4132583  PMID: 24731132
blood–brain barrier; hypothermia; inflammation; neurotensin analogue; sensorimotor function; traumatic brain injury
15.  Levels of Urbanization and Parental Education in Relation to the Mortality Risk of Young Children 
Background: The establishment of the National Health Insurance program in Taiwan in 1995 effectively removed the financial barrier to access health care services of Taiwanese people. This population-based cohort study aimed to determine the independent and joint effects of parental education and area urbanization on the mortality risk among children under the universal health insurance coverage in Taiwan since 1995. Methods: We linked 1,501,620 births from 1996 to 2000 to the Taiwan Death Registry to estimate the neonatal, infant, and under-five mortality rates, according to the levels of parental education and urbanization of residential areas. We used a logistic regression model that considers data clustering to estimate the independent and joint effects. Results: Lower levels of parental education and area urbanization exerted an independent effect of mortality on young children, with a stronger magnitude noted for areas with lower levels of urbanization. Children whose parents had lower levels of education and who were born in areas with lower levels of urbanization experienced the highest risk for neonatal (odds ratio (OR) = 1.60, 95% CI = 1.46–1.76), infant (OR = 1.58, 95% CI = 1.48–1.70), and under-five (OR = 1.71, 95% CI = 1.61–1.82) mortality. Conclusions: Even with universal health insurance coverage, lower levels of area urbanization and parental education still exerted independent and joint effects on mortality in young children. This finding implies the inadequate accessibility to health care resources for children from socially disadvantaged families and less urbanized areas.
PMCID: PMC4515684  PMID: 26184248
neonatal mortality; infant mortality; under-five mortality; socioeconomic status; cohort studies
16.  Derivation of Cinnamon Blocks Leukocyte Attachment by Interacting with Sialosides 
PLoS ONE  2015;10(6):e0130389.
Molecules derived from cinnamon have demonstrated diverse pharmacological activities against infectious pathogens, diabetes and inflammatory diseases. This study aims to evaluate the effect of the cinnamon-derived molecule IND02 on the adhesion of leukocytes to host cells. The anti-inflammatory ability of IND02, a pentameric procyanidin type A polyphenol polymer isolated from cinnamon alcohol extract, was examined. Pretreatment with IND02 significantly reduced the attachment of THP-1 cells or neutrophils to TNF-α-activated HUVECs or E-selectin/ICAM-1, respectively. IND02 also reduced the binding of E-, L- and P-selectins with sialosides. Furthermore, IND02 could agglutinate human red blood cells (RBC), and the agglutination could be disrupted by sialylated glycoprotein. Our findings demonstrate that IND02, a cinnamon-derived compound, can interact with sialosides and block the binding of selectins and leukocytes with sialic acids.
PMCID: PMC4468131  PMID: 26076445
17.  Identification of differentially expressed signatures of long non-coding RNAs associated with different metastatic potentials in gastric cancer 
Journal of Gastroenterology  2015;51:119-129.
Gastric cancer (GC) is known for its lymph node metastasis and outstanding morbidity and mortality. Thus, improvement in the current knowledge regarding the molecular mechanism of GC is urgently needed to discover novel biomarkers involved in its progression and prognosis. Several long, non-coding RNAs (lncRNAs) play important roles in gastric tumorigenesis and metastasis. However, the signature of lncRNA-associated metastasis in GC is not fully clarified.
We determined the lncRNA and mRNA expression profiles correlating to GC with or without lymph node-metastasis based on microarray analysis. Twelve differentially expressed lncRNAs and six differentially expressed mRNAs were validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay.
The relationships between the aberrantly expressed lncRNAs XLOC_010235 or RP11-789C1.1 and lymph node metastasis, pathologic metastasis status, distal metastasis and TNM (tumour, node, and metastasis) stage were found to be significantly different. Via survival analysis, patients who had high-expressed XLOC_010235 or low-expressed RP11-789C1.1 showed significantly worse survival than patients with inverse-expressed XLOC_010235 or RP11-789C1.1.
In summary, this current study highlights some evidence regarding the potential role of lncRNAs in GC and posits that specific lncRNAs can be identified as novel, poor prognostic biomarkers in GC.
Electronic supplementary material
The online version of this article (doi:10.1007/s00535-015-1091-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4742487  PMID: 26045391
GC; Metastasis; LncRNA; Microarray; Biomarker
18.  High-dose chemotherapy combined with autologous peripheral blood stem cell transplantation in children with advanced malignant solid tumors: A retrospective analysis of 38 cases 
Oncology Letters  2015;10(2):1047-1053.
The aim of the present study was to assess the toxicity and efficacy of autologous peripheral blood stem cell (APBSC) transplantation in children with advanced malignant solid tumors. The outcomes of 38 children with advanced malignant solid tumor, who were treated with high-dose chemotherapy and autologous peripheral blood stem cell transplantation in Beijing Tongren Hospital (Capital Medical University, Beijing, China) between September 2005 and November 2011, were retrospectively analyzed. The effects of treatment were evaluated according to the standard Bearman's criteria. The mean count of collected mononuclear cells and the cluster of differentiation 34+ cell count from 38 patients was 5.6±2.2×108/kg and 3.8±2.6×106/kg, respectively. From these 38 patients, the number of stem cells collected from 31 cases (81.6%) accorded with the transplantation standards. Three and 14 days after pretreatment in these 38 cases, there were 19 cases of grade I, 11 cases of grade II, five cases of grade III and three cases of grade IV (one case succumbed) adverse reaction. Following the treatment (23–40 days after pretreatment, during organ injury recovery), 37 cases obtained bone marrow reconstitution with a mean time of 12.3±3.1 days after APBSC reinfusion. The median survival time of the 37 patients was 49 months, and the survival rate at one, three and five years post-treatment was 91.9, 68.2 and 36.6%, respectively.
PMCID: PMC4508994  PMID: 26622624
autologous; peripheral blood stem cell; transplantation; solid tumor; children
19.  Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling 
Stem Cells International  2015;2015:105172.
Human pluripotent stem cells (hPSCs) derived from either blastocyst stage embryos (hESCs) or reprogrammed somatic cells (iPSCs) can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as for in vitro modeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulate in vivo conditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs.
PMCID: PMC4454762  PMID: 26089911
20.  Mortality of Combined Serum Phosphorus and Parathyroid Hormone Concentrations and their Changes over Time in Hemodialysis Patients 
Bone  2014;61:201-207.
Mineral and bone disorder (MBD) is common and associated with mortality in patients with chronic kidney disease (CKD). Given that disarrays in serum phosphorus (P) and parathyroid hormone (PTH) levels and their changes over time are closely interrelated, modeling mortality-predictability of their combinations may help improve CKD patient management.
A historical cohort study was undertaken to evaluate the joint effect of serum P and PTH levels on mortality in 107,299 chronic hemodialysis (HD) patients. Changes in serum P and PTH levels over 6 months, in particular discordant changes, were also modeled with mortality.
HD patients were 64±15 (mean±SD) years old and included 45% women, 33% African–American, and 59% diabetic. Compared with serum P level ≥7.0 mg/dL and PTH level ≥600 pg/mL, adjusted hazard ratio (HR) tended to be lowest in patients with serum P level of 3.5–<5.5 mg/dL combined with PTH level of 150–<300 pg/mL (HR 0.64, 95% confidence interval 0.61–0.67). A change over time in serum P level towards the 3.5–<5.5 mg/dL range from higher or lower ranges was associated with a decreased mortality, whereas only change in PTH level from <150 pg/mL to 150–<300 pg/mL range was associated with a lower risk of mortality. Upon discordant changes of PTH and P, i.e., decrease in one of the two measures while the other increased, no change in mortality risk was observed.
In CKD–MBD management, patent survival is the greatest with controlling both serum P and PTH levels in balance. Tailoring an individualized treatment strategy in CKD-MBD may benefit patients. Further studies are needed.
PMCID: PMC4024455  PMID: 24486956
bone; hemodialysis; hyperparathyroidism; mineral; phosphorus
21.  Lewisy Promotes Migration of Oral Cancer Cells by Glycosylation of Epidermal Growth Factor Receptor 
PLoS ONE  2015;10(3):e0120162.
Aberrant glycosylation changes normal cellular functions and represents a specific hallmark of cancer. Lewisy (Ley) carbohydrate upregulation has been reported in a variety of cancers, including oral squamous cell carcinoma (OSCC). A high level of Ley expression is related to poor prognosis of patients with oral cancer. However, it is unclear how Ley mediates oral cancer progression. In this study, the role of Ley in OSCC was explored. Our data showed that Ley was upregulated in HSC-3 and OC-2 OSCC cell lines. Particularly, glycosylation of epidermal growth factor receptor (EGFR) with Ley was found in OC-2 cells, and this modification was absent upon inhibition of Ley synthesis. The absence of Ley glycosylation of EGFR weakened phosphorylation of AKT and ERK in response to epidermal growth factor (EGF). Additionally, EGF-triggered cell migration was reduced, but cell proliferation was not affected. Ley modification stabilized EGFR upon ligand activation. Conversely, absence of Ley glycosylation accelerated EGFR degradation. In summary, these results indicate that increased expression of Ley in OSCC cells is able to promote cell migration by modifying EGFR which in turn stabilizes EGFR expression and downstream signaling. Targeting Ley on EGFR could have a potential therapeutic effect on oral cancer.
PMCID: PMC4370659  PMID: 25799278
22.  Association between Thiopurine S-methyltransferase Polymorphisms and Thiopurine-Induced Adverse Drug Reactions in Patients with Inflammatory Bowel Disease: A Meta-Analysis 
PLoS ONE  2015;10(3):e0121745.
Thiopurine drugs are well established treatments in the management of inflammatory bowel disease (IBD), but their use is limited by significant adverse drug reactions (ADRs). Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in thiopurine metabolism. Several clinical guidelines recommend determining TPMT genotype or phenotype before initiating thiopurine therapy. Although several studies have investigated the association between TPMT polymorphisms and thiopurine-induced ADRs, the results are inconsistent. The purpose of this study is to evaluate whether there is an association between TPMT polymorphisms and thiopurine-induced ADRs using meta-analysis.
We explored PubMed, Web of Science and Embase for articles on TPMT polymorphisms and thiopurine-induced ADRs. Studies that compared TPMT polymorphisms with-ADRs and without-ADRs in IBD patients were included. Relevant outcome data from all the included articles were extracted and the pooled odds ratio (OR) with corresponding 95% confidence intervals were calculated using Revman 5.3 software.
Fourteen published studies, with a total of 2,206 IBD patients, which investigated associations between TPMT polymorphisms and thiopurine-induced ADRs were included this meta-analysis. Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with thiopurine-induced overall ADRs and bone marrow toxicity; pooled ORs were 3.36 (95%CI: 1.82–6.19) and 6.67 (95%CI: 3.88–11.47), respectively. TPMT polymorphisms were not associated with the development of other ADRs including hepatotoxicity, pancreatitis, gastric intolerance, flu-like symptoms and skin reactions; the corresponding pooled ORs were 1.27 (95%CI: 0.60–2.71), 0.97 (95%CI: 0.38–2.48), 1.82 (95%CI: 0.93–3.53), 1.28 (95%CI: 0.47–3.46) and 2.32 (95%CI: 0.86–6.25), respectively.
Our meta-analysis demonstrated an association of TPMT polymorphisms with overall thiopurine-induced ADRs and bone marrow toxicity, but not with hepatotoxicity, pancreatitis, flu-like symptoms, gastric intolerance and skin reactions. These findings suggest that pretesting the TPMT genotype could be helpful in clinical practice before initiating thiopurine therapy. However, white blood cell count analysis should be the mainstay for follow-up.
PMCID: PMC4370632  PMID: 25799415
23.  Identification and Characterization of Two Novel Spliced Genes Located in the orf47-orf46-orf45 Gene Locus of Kaposi's Sarcoma-Associated Herpesvirus 
Journal of Virology  2014;88(17):10092-10109.
The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino-acid motif, and the complete tegument protein ORF45. Like the ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at the endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation, and knockdown of GRP78 in cells significantly impairs viral lytic cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through the ER, our results strongly implicate that the ORF47/45 proteins may serve as key effectors for controlling GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle.
IMPORTANCE Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. The two parts of this study identify two aspects of the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part characterizes the function of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein, GRP78. In addition to the ability to promote GRP78 upregulation, the ORF47/45 proteins also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of GRP78 expression by viral proteins at the early lytic stage may aid with the protection of host cells from severe ER stress and may directly involve the assembly or release of virions.
PMCID: PMC4136336  PMID: 24965448
24.  Boronophenylalanine, a boron delivery agent for boron neutron capture therapy, is transported by ATB0,+, LAT1 and LAT2 
Cancer Science  2015;106(3):279-286.
The efficacy of boron neutron capture therapy relies on the selective delivery of boron carriers to malignant cells. p-Boronophenylalanine (BPA), a boron delivery agent, has been proposed to be localized to cells through transporter-mediated mechanisms. In this study, we screened aromatic amino acid transporters to identify BPA transporters. Human aromatic amino acid transporters were functionally expressed in Xenopus oocytes and examined for BPA uptake and kinetic parameters. The roles of the transporters in BPA uptake were characterized in cancer cell lines. For the quantitative assessment of BPA uptake, HPLC was used throughout the study. Among aromatic amino acid transporters, ATB0,+, LAT1 and LAT2 were found to transport BPA with Km values of 137.4 ± 11.7, 20.3 ± 0.8 and 88.3 ± 5.6 μM, respectively. Uptake experiments in cancer cell lines revealed that the LAT1 protein amount was the major determinant of BPA uptake at 100 μM, whereas the contribution of ATB0,+ became significant at 1000 μM, accounting for 20–25% of the total BPA uptake in MCF-7 breast cancer cells. ATB0,+, LAT1 and LAT2 transport BPA at affinities comparable with their endogenous substrates, suggesting that they could mediate effective BPA uptake in vivo. The high and low affinities of LAT1 and ATB0,+, respectively, differentiate their roles in BPA uptake. ATB0,+, as well as LAT1, could contribute significantly to the tumor accumulation of BPA at clinical dose.
PMCID: PMC4376436  PMID: 25580517
Amino acid transporter; BNCT; BPA; cancer cell lines; drug delivery
25.  Ubiquitin-Binding Site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23 
Nature communications  2014;5:4638.
Polyglutamine repeat expansion in ataxin-3 causes neurodegeneration in the most common dominant ataxia, Spinocerebellar Ataxia Type 3 (SCA3). Since reducing levels of disease proteins improves pathology in animals, we investigated how ataxin-3 is degraded. Here we show that, unlike most proteins, ataxin-3 turnover does not require its ubiquitination, but is regulated by Ubiquitin-Binding Site 2 (UbS2) on its N terminus. Mutating UbS2 decreases ataxin-3 protein levels in cultured mammalian cells and in Drosophila melanogaster by increasing its proteasomal turnover. Ataxin-3 interacts with the proteasome-associated proteins Rad23A/B through UbS2. Knockdown of Rad23 in cultured cells and in Drosophila results in lower levels of ataxin-3 protein. Importantly, reducing Rad23 suppresses ataxin-3-dependent degeneration in flies. We present a mechanism for ubiquitination-independent degradation that is impeded by protein interactions with proteasome-associated factors. We conclude that UbS2 is a potential target through which to enhance ataxin-3 degradation for SCA3 therapy.
PMCID: PMC4237202  PMID: 25144244
Deubiquitinase; Drosophila; Neurodegeneration; Neuroprotection; Polyglutamine; Proteasome; hHR23; Spinocerebellar Ataxia; Ubiquitin

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