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1.  Hypoxia augments the calcium-activated chloride current carried by anoctamin-1 in cardiac vascular endothelial cells of neonatal mice 
British Journal of Pharmacology  2014;171(15):3680-3692.
The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs).
Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs.
Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl(Ca) detected in CVECs was significantly inhibited by T16Ainh-A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl(Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl(Ca) was inhibited by T16Ainh-A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl(Ca) in Ano1 gene knockdown CVECs.
Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl(Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca2+. Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage.
PMCID: PMC4128065  PMID: 24758567
calcium-activated chloride channels; cardiac vascular endothelial cells; anoctamin; hypoxia
2.  Practical Use of Bone Scan in Patients with an Osteoporotic Vertebral Compression Fracture 
Journal of Korean Medical Science  2015;30(2):194-198.
Rib fractures are one of main causes of chest or flank pain when related to an osteoporotic vertebral compression fracture (OVCF). The authors investigated the incidence and risk factors of rib fracture in 284 patients with OVCF using bone scans and evaluated the feasibility as to whether bone scans could be utilized as a useful screening tool. Hot uptake lesions on ribs were found in 122 cases (43.0%). The factors analyzed were age, sex, number and locations of fractured vertebrae, BMD, and compression rates as determined using initial radiography. However, no statistical significances were found. In 16 cases (5.6%), there were concurrent multiple fractures of both the thoracic and lumbar spines not detected by single site MRI. Sixty cases (21.1%) of OVCF with the a compression rate of less than 15% could not be identified definitely by initial plain radiography, but were confirmed by bone scans. It is concluded that a bone scan has outstanding ability for the screening of rib fractures associated with OVCF. Non-adjacent multiple fractures in both thoracic and lumbar spines and fractures not identified definitely by plain radiography were detected on bone scans, which provided a means for determining management strategies and predicting prognosis.
Graphical Abstract
PMCID: PMC4310947  PMID: 25653492
Osteoporosis; Spine; Compression Fracture; Rib Fracture; Osteoporotic Fracture
3.  Hexavalent chromium in house dust — A comparison between an area with historic contamination from chromate production and background locations 
The Science of the total environment  2010;408(21):4993-4998.
In contrast to Cr+ 3, Cr+ 6 is carcinogenic and allergenic. Although Cr+ 6 can occur naturally, it is thought that most soil Cr+ 6 is anthropogenic, however, the extent of Cr+ 6 in the background environment is unknown. Cr+ 6-containing chromite ore processing residue (COPR) from chromate manufacture was deposited in numerous locations in Jersey City (JC), New Jersey. In the 1990’s, significantly elevated concentrations of total Cr (Cr+ 6+Cr+ 3) were found in house dust near COPR sites. We undertook a follow-up study to determine ongoing COPR exposure. We compared Cr+6 in house dust in JC to selected background communities with no known sources of Cr+ 6. Samples were collected from living areas, basements and window wells. Cr+6 was detected in dust from all JC and background houses. In the JC homes, the mean (±SD) Cr+ 6 concentration for all samples was 3.9±7.0 μg/g (range: non-detect–90.4 μg/g), and the mean Cr+ 6 loading was 5.8±15.7 μg/m2 (range: non-detect–196.4 μg/m2). In background homes, the mean Cr+ 6 concentrations of all samples was 4.6±7.8 μ μg/g, (range, 0.05–56.6 μg/g). The mean loading was 10.0±27.9 μg/m2 (range, 0.22–169.3 μg/m2). There was no significant difference between Cr+ 6 dust concentrations in Jersey City and background locations. Stratification by sample location within houses and sampling method gave similar results. Samples exceeding 20 μg/g were obtained only from single wood surfaces in different homes. Lower concentrations in window well samples suggests transport from outside is not the major source of indoor Cr+ 6. Landscaping and groundcover may influence indoor Cr+6. There appears to be a widespread low level background of Cr+ 6 that is not elevated in Jersey City homes despite its historic COPR contamination. It is possible that house dust, in general, is a source of Cr+ 6 exposure with potential implications for persistence of chromium allergic contact dermatitis.
PMCID: PMC4300124  PMID: 20692023
Chromium; Hexavalent chromium; Cr+6; Dust; Dust loading; COPR; Chromate; Exposure assessment; Chromium waste
4.  Activation of the Ubiquitin Proteasome Pathway by Silk Fibroin Modified Chitosan Nanoparticles in Hepatic Cancer Cells 
Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 μg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation.
PMCID: PMC4307326  PMID: 25588218
silk fibroin; chitosan nanoparticle; biomaterial; proteomics; ubiquitin proteasome pathway
5.  Epigenetic signature and enhancer activity of the human APOE gene 
Human Molecular Genetics  2013;22(24):5036-5047.
The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ɛ2/ɛ3/ɛ4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ɛ4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ɛ2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ɛ2/ɛ3/ɛ4 allele-carrying 3′-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ɛ2/ɛ3/ɛ4 allele-specific manner. These findings implicate a novel functional role for a 3′-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI.
PMCID: PMC3836480  PMID: 23892237
6.  Anoxic Androgen Degradation by the Denitrifying Bacterium Sterolibacterium denitrificans via the 2,3-seco Pathway 
Applied and Environmental Microbiology  2014;80(11):3442-3452.
The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C19 androgens. By using a 13C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the identification of a C17 intermediate, we propose that the A-ring cleavage may be followed by the removal of a C2 side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (≤500 μM) did not exhibit androgenic activity, whereas the sterane-containing intermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteobacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, and both of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acid can be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C19 androgens.
PMCID: PMC4018845  PMID: 24657867
7.  Characterization of Exoelectrogenic Bacteria Enterobacter Strains Isolated from a Microbial Fuel Cell Exposed to Copper Shock Load 
PLoS ONE  2014;9(11):e113379.
Microorganisms capable of generating electricity in microbial fuel cells (MFCs) have gained increasing interest. Here fourteen exoelectrogenic bacterial strains were isolated from the anodic biofilm in an MFC before and after copper (Cu) shock load by Hungate roll-tube technique with solid ferric (III) oxide as an electron acceptor and acetate as an electron donor. Phylogenetic analysis of the 16S rRNA gene sequences revealed that they were all closely related to Enterobacter ludwigii DSM 16688T within the Enterobacteriaceae family, although these isolated bacteria showed slightly different morphology before and after Cu shock load. Two representative strains R2B1 (before Cu shock load) and B4B2 (after Cu shock load) were chosen for further analysis. B4B2 is resistant to 200 mg L−1 of Cu(II) while R2B1 is not, which indicated the potential selection of the Cu shock load. Raman analysis revealed that both R2B1 and B4B2 contained c-type cytochromes. Cyclic voltammetry measurements revealed that strain R2B1 had the capacity to transfer electrons to electrodes. The experimental results demonstrated that strain R2B1 was capable of utilizing a wide range of substrates, including Luria-Bertani (LB) broth, cellulose, acetate, citrate, glucose, sucrose, glycerol and lactose to generate electricity, with the highest current density of 440 mA·m−2 generated from LB-fed MFC. Further experiments indicated that the bacterial cell density had potential correlation with the current density.
PMCID: PMC4239067  PMID: 25412475
8.  Structural Analysis of Cytochrome bc1 Complexes: Implications to the Mechanism of Function 
Biochimica et biophysica acta  2012;1827(0):1278-1294.
The cytochrome bc1 complex (bc1) is the mid-segment of the cellular respiratory chain of mitochondria and many aerobic prokaryotic organisms; it is also part of the photosynthetic apparatus of non-oxygenic purple bacteria. The bc1 complex catalyzes the reaction of transferring electrons from the low potential substrate ubiquinol to high potential cytochrome c. Concomitantly, bc1 translocates protons across the membrane, contributing to the proton-motive force essential for a variety of cellular activities such as ATP synthesis. Structural investigations of bc1 have been exceedingly successful, yielding atomic resolution structures of bc1 from various organisms and trapped in different reaction intermediates. These structures have confirmed and unified results of decades of experiments and have contributed to our understanding of the mechanism of bc1 functions as well as its inactivation by respiratory inhibitors.
PMCID: PMC3593749  PMID: 23201476
cytochrome bc1 complex; bifurcated electron flow; mechanism of ubiquinol oxidation; crystal structure; control of ISP domain movement
9.  An LC-MS assay for the screening of cardiovascular medications in human samples 
Cardiovascular drugs are the most commonly prescribed medications. Some prior assays successfully detect cardiovascular drugs among multiple classes using a single sample. Here, we develop an assay to detect a broad range of cardiovascular drug classes to include commonly used cardiovascular drugs and evaluate the assay’s analytical and statistical properties in a clinical setting. We describe a protocol for drug detection that encompasses 34 commonly prescribed cardiovascular drugs or drug metabolites with a single LC-MS/MS method using 100µl of serum or plasma. Drug classes monitored by this assay include: anticoagulants, angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB), beta blockers, calcium channel blockers, diuretics, statins, and vasodilators, as well as digoxin, fenofibrate, and niacin.
Analytical accuracy and precision for each drug was evaluated by repeating the assay on spiked samples at low, medium, and high concentrations. In 294 clinical samples obtained from hospitalized patients for whom medication administration was recorded, we evaluated the assay’s statistical sensitivity, specificity, and accuracy. For the 34 drugs or drug metabolites, the assay was statistically sensitive (>0.90) for all drugs except captopril (0.25), isosorbide (0.81), and niacin (0.89). The assay was statistically specific for all drugs, with a minimum specificity of 0.94 (aspirin). To our knowledge, this method is the first method of simultaneous analysis of 34 cardiovascular drugs or drug metabolites from nine drug classes evaluated using clinical samples from hospitalized patients.
PMCID: PMC3800555  PMID: 24013190
cardiovascular drugs; drug monitoring; selectivity; mass spectrometry; liquid chromatography; clinical samples
10.  Do low-income coronary artery bypass surgery patients have equal opportunity to access excellent quality of care and enjoy good outcome in Taiwan? 
Equity is an important issue in the healthcare research field. Many studies have focused on the relationship between patient characteristics and outcomes of care. These studies, however, have seldom examined whether patients’ characteristics affected their access to quality healthcare, which further affected the care outcome. The purposes of this study were to determine whether low-income coronary artery bypass surgery (CABG) patients receive healthcare services with poorer quality, and if such differences in treatment result in different outcomes.
A retrospective multilevel study design was conducted using claims data from Taiwan’s universal health insurance scheme for 2005-2008. Patients who underwent their CABG surgery between 2006 and 2008 were included in this study. CABG patients who were under 18 years of age or had unknown gender or insured classifications were excluded. Hospital and surgeon’s performance indicators in the previous one year were used to evaluate the level of quality via k-means clustering algorithm. Baron and Kenny’s procedures for mediation effect were conducted to explore the relationship among patient’s income, quality of CABG care, and inpatient mortality.
A total of 10,320 patients were included in the study. The results showed that 5.65% of the low-income patients received excellent quality of care, which was lower than that of patients not in the low-income group (5.65% vs.11.48%). The mortality rate of low-income patients (12.10%) was also higher than patients not in the low-income group (5.25%). Also, the mortality of patients who received excellent care was half as low as patients receiving non-excellent care (2.63% vs. 5.68%). Finally, after the procedure of mediation effect testing, the results showed that the relationship between patient income level and CABG mortality was partially mediated by patterns of quality of care.
The results of the current study implied that worse outcome in low-income CABG patients might be associated with poorer quality of received services. Health authorities should pay attention to this issue, and propose appropriate solutions.
PMCID: PMC4159514  PMID: 25052723
Quality of care; Health inequity; Patterns of care; CABG
11.  TOMM40 intron 6 poly-T length, age-at-onset and neuropathology of AD in individuals with APOE ε3/ε3 
This study investigates the association between TOMM40 poly-T length, age-at-onset, and neuropathology in Alzheimer’s disease (AD) individuals with the APOE ε3/ε3 allele.
Thirty-two PSEN1 mutation carriers with AD, 27 PSEN2 mutation carriers with AD, 59 participants with late-onset AD (LOAD), and 168 participants with autopsies from a community-based cohort were genotyped for TOMM40 intron 6 poly-T (rs10524523) length using short tandem repeat assays.
Among AD patients with PSEN2 mutations, the presence of a long poly-T was associated with an earlier age-at-onset, whereas there were no such associations for patients with PSEN1 mutations or LOAD. In community-based participants, the presence of a long poly-T was associated with increased neuritic tangles and a higher likelihood of pathologically diagnosed AD.
TOMM40 intron 6 poly-T length may explain some of the variation in age-at-onset in PSEN2 familial AD and may be associated with AD neuropathology in persons with APOE ε3/ε3.
PMCID: PMC3606272  PMID: 23183136
Alzheimer’s disease; age-at-onset; genetic; APOE; TOMM40; PSEN1 mutation; PSEN2 mutation; neuropathology
12.  Pollen Count and Presentation of Angiotensin-Converting Enzyme Inhibitor-Associated Angioedema 
The incidence of angiotensin-converting enzyme (ACE) inhibitor-associated angioedema is increased in patients with seasonal allergies.
We tested the hypothesis that patients with ACE inhibitor-associated angioedema present during months when pollen counts are increased.
Cohort analysis examined the month of presentation of ACE inhibitor-associated angioedema and pollen counts in the ambulatory and hospital setting. Patients with ACE inhibitor-associated angioedema were ascertained through (1) an observational study of patients presenting to Vanderbilt University Medical Center, (2) patients presenting to the Marshfield Clinic and participating in the Marshfield Clinic Personalized Medicine Research Project, and (3) patients enrolled in The Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial (ONTARGET). Measurements include date of presentation of ACE inhibitor-associated angioedema, population exposure to ACE inhibitor by date, and local pollen counts by date.
At Vanderbilt, the rate of angioedema was significantly associated with tree pollen months (P = .01 from χ2 test). When separate analyses were conducted in patients with a history of seasonal allergies and patients without, the rate of ACE inhibitor-associated angioedema was increased during tree pollen months only in patients with a history of seasonal allergies (P = .002). In Marshfield, the rate of angioedema was significantly associated with ragweed pollen months (P = .025). In ONTARGET, a positive trend was observed between the ACE inhibitor-associated angioedema rate and grass season, although it was not statistically significant (P = .057).
Patients with ACE inhibitor-associated angioedema are more likely to present with this adverse drug event during months when pollen counts are increased.
PMCID: PMC4042396  PMID: 24565618
Angiotensin-converting enzyme inhibitor; angioedema; pollen; bradykinin; substance P; seasonal allergies
13.  Influence of seasonal exposure to grass pollen on local and peripheral blood IgE repertoires in patients with allergic rhinitis 
Previous studies of immunoglobulin gene sequences in patients with allergic diseases using low-throughput Sanger sequencing have limited the analytic depth for characterization of IgE repertoires.
We used a high-throughput, next-generation sequencing approach to characterize immunoglobulin heavy-chain gene (IGH) repertoires in patients with seasonal allergic rhinitis (AR) with the aim of better understanding the underlying disease mechanisms.
IGH sequences in matched peripheral blood and nasal biopsy specimens from nonallergic healthy control subjects (n = 3) and patients with grass pollen–related AR taken in season (n = 3) or out of season (n = 4) were amplified and pyrosequenced on the 454 GS FLX+ System.
A total of 97,610 IGH (including 8,135 IgE) sequences were analyzed. Use of immunoglobulin heavy-chain variable region gene families 1 (IGHV1) and 5 (IGHV5) was higher in IgE clonotypic repertoires compared with other antibody classes independent of atopic status. IgE repertoires measured inside the grass pollen season were more diverse and more mutated (particularly in the biopsy specimens) and had more evidence of antigen-driven selection compared with those taken outside of the pollen season or from healthy control subjects. Clonal relatedness was observed for IgE between the blood and nasal biopsy specimens. Furthermore in patients with AR, but not healthy control subjects, we found clonal relatedness between IgE and IgG classes.
This is the first report that exploits next-generation sequencing to determine local and peripheral blood IGH repertoires in patients with respiratory allergic disease. We demonstrate that natural pollen exposure was associated with changes in IgE repertoires that were suggestive of ongoing germinal center reactions. Furthermore, these changes were more often apparent in nasal biopsy specimens compared with peripheral blood and in patients with AR compared with healthy control subjects.
PMCID: PMC4151999  PMID: 25171866
Next-generation sequencing; peripheral blood and nasal mucosal IgE repertoires; allergic rhinitis; AR, Allergic rhinitis; AR.IS, Allergic rhinitis inside the pollen season; AR.OS, Allergic rhinitis outside the pollen season; CDR, Complementarity-determining region; CSR, Class-switch recombination; GC, Germinal center; NA, Nonallergic healthy control subject; NGS, Next-generation sequencing; QC, Quality control; SHM, Somatic hypermutation
14.  Changes in endotracheal tube cuff pressure during laparoscopic surgery in head-up or head-down position 
BMC Anesthesiology  2014;14:75.
The abdominal insufflation and surgical positioning in the laparoscopic surgery have been reported to result in an increase of airway pressure. However, associated effects on changes of endotracheal tube cuff pressure are not well established.
70 patients undergoing elective laparoscopic colorectal tumor resection (head-down position, n = 38) and laparoscopic cholecystecomy (head-up position, n = 32) were enrolled and were compared to 15 patients undergoing elective open abdominal surgery. Changes of cuff and airway pressures before and after abdominal insufflation in supine position and after head-down or head-up positioning were analysed and compared.
There was no significant cuff and airway pressure changes during the first fifteen minutes in open abdominal surgery. After insufflation, the cuff pressure increased from 26 ± 3 to 32 ± 6 and 27 ± 3 to 33 ± 5 cmH2O in patients receiving laparoscopic cholecystecomy and laparoscopic colorectal tumor resection respectively (both p < 0.001). The head-down tilt further increased cuff pressure from 33 ± 5 to 35 ± 5 cmH2O (p < 0.001). There six patients undergoing colorectal tumor resection (18.8%) and eight patients undergoing cholecystecomy (21.1%) had a total increase of cuff pressure more than 10 cm H2O (18.8%). There was no significant correlation between increase of cuff pressure and either the patient's body mass index or the common range of intra-abdominal pressure (10-15 mmHg) used in laparoscopic surgery.
An increase of endotracheal tube cuff pressure may occur during laparoscopic surgery especially in the head-down position.
PMCID: PMC4160323  PMID: 25210501
Endotracheal tube cuff pressure; Laparoscopic surgery; Head-down position; Head-up position
15.  Ligand-activated PPAR-γ protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3ε upregulation 
Circulation  2009;119(8):1124-1134.
Thiazolidinediones (TZD) were reported to protect against ischemia-reperfusion (I/R) injury. Their protective actions are considered to be PPAR-γ (peroxisome proliferator-activated receptor γ)-dependent. However, it is unclear how PPAR-γ activation confers resistance to I/R.
Methods and Results
We evaluated the effects of rosiglitazone or PPAR-γ overexpression on cerebral infarction in a rat model and investigated the anti-apoptotic actions in N2-A neuroblastoma cell model. Rosiglitazone or PPAR-γ overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-γ siRNA. In mice with knockin of a PPAR-γ domain negative mutant, infarct volume was enhanced. Proteomic analysis reveals that brain 14-3-3ε was highly upregulated in rats treated with rosiglitazone. 14-3-3ε upregulation was abrogated by PPAR-γ siRNA or antagonist. Promoter analysis and chromatin immunoprecipitation reveal that rosiglitazone induced PPAR-γ binding to specific regulatory elements on 14-3-3ε promoter and thereby increased 14-3-3ε transcription. 14-3-3ε siRNA abrogated the anti-apoptotic actions of rosiglitazone or PPAR-γ overexpression while 14-3-3ε recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3ε enhanced binding of phosphorylated Bad, and protected mitochondrial membrane potential.
Ligand-activated PPAR-γ confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3ε transcription. 14-3-3ε upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.
PMCID: PMC4144045  PMID: 19221220
Apoptosis; Infarction; Stroke; PPAR; 14-3-3ε
16.  The Contribution of Antibiotic Resistance Mechanisms in Clinical Burkholderia cepacia Complex Isolates: An Emphasis on Efflux Pump Activity 
PLoS ONE  2014;9(8):e104986.
Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.
PMCID: PMC4143217  PMID: 25153194
17.  Evaluation of the Antioxidant Activity and Antiproliferative Effect of the Jaboticaba (Myrciaria cauliflora) Seed Extracts in Oral Carcinoma Cells 
BioMed Research International  2014;2014:185946.
It is becoming increasingly evident that certain phytochemicals possess cancer chemopreventive properties. In this study, the antiproliferative activity of extracts from different parts of the jaboticaba (Myrciaria cauliflora) plant was evaluated for its effect on human oral carcinoma cell lines. The cytotoxicities of various plant extract concentrations were examined and the 50% maximal inhibitory concentration (IC50) was determined. Water extracts of jaboticaba seeds showed concentration-dependent antiproliferative effects. Annexin V/propidium iodide positivity with active caspase-3 induction indicated that the treated cells underwent apoptosis. Several important regulatory proteins (Bcl-2, Bcl-xL, Bid, and survivin) involved in apoptosis were also evaluated. The antioxidant activity of jaboticaba was investigated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, and the drug concentration eliciting 50% maximum stimulation (SC50) was determined. The present findings suggest that water extracts of jaboticaba seeds exhibit an antiproliferative effect against oral cancer cells by inducing apoptosis through downregulating survivin expression and thereby activating caspase-mediated Bid cleavage.
PMCID: PMC4150497  PMID: 25197631
18.  Co-activation of ERK, NF-κB, and GADD45β in Response to Ionizing Radiation* 
The Journal of biological chemistry  2005;280(13):12593-12601.
NF-κB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45β is a primary cell cycle element responsive to NF-κB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-κB, ERK and GADD45β are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-κB transactivation and GADD45β expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-κB. Transfection of dominant negative mutant IκB that blocks NF-κB nuclear translocation, inhibited ERK activity and GADD45β expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Anti-sense inhibition of GADD45β strikingly blocked IR-induced NF-κB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-κB, ERK, and GADD45β are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.
PMCID: PMC4130153  PMID: 15642734
19.  Toxocara Seroprevalence among Clinically Healthy Individuals, Pregnant Women and Psychiatric Patients and Associated Risk Factors in Shandong Province, Eastern China 
Toxocarosis is a widespread zoonosis caused by the ascarid nematodes Toxocara canis and Toxocara cati, which primarily infect dogs and cats, respectively. Most human infections with Toxocara are asymptomatic; however, some infected individuals may develop a serious illness and even death. Nevertheless, epidemiological knowledge regarding the prevalence and risks associated with Toxocara infection is limited in China. Therefore, we performed a cross-sectional pilot study and estimated the seroprevalence of Toxocara infection in humans in Shandong Province, eastern China for the first time, from June 2011 to July 2013, involving clinically healthy individuals, pregnant women and psychiatric patients, aiming to attract public attention to Toxocara infection.
Methodology/Principle Findings
Seroprevalence of Toxocara was determined using an enzyme-linked immunosorbent assay based on a cross-sectional study conducted in Qingdao and Weihai, Shandong Province, eastern China. Factors potentially associated with Toxocara infection were identified by logistic regression analysis. The overall Toxocara seroprevalence among the study population (n = 2866) was 12.25%, and a significantly higher seroprevalence in psychiatric patients (16.40%, 73/445) than that in clinically healthy individuals (13.07%, 187/1431) and pregnant women (9.19%, 91/990) was revealed. Univariate analyses suggested that keeping dogs at home (OR = 0.06, 95% CI 0.05–0.08, P<0.001), contact with cats and dogs (OR = 0.42, 95% CI 0.33–0.53, P<0.001) and exposure with soil (OR = 0.37, 95% CI 0.28–0.49, P<0.001) were risk factors associated with Toxocara infection.
The present study revealed, for the first time, that human infection with Toxocara is common in eastern China, posing a significant public health concern. Increasing human and dog populations, population movements and climate change all will serve to increase the importance of this zoonosis. Further studies under controlled conditions are necessary to define potential morbidity associated with Toxocara infection.
Author Summary
Toxocarosis, a typical neglected and underestimated human health problem, is caused by the ascarid nematodes Toxocara canis and Toxocara cati, which primarily infect dogs and cats, respectively. Previous studies have reported an increased risk for Toxocara infection in humans worldwide, especially in children and psychiatric patients. This pilot study was aimed to investigate the Toxocara serology in clinically healthy individuals, pregnant women and psychiatric patients in Shandong Province, eastern China using an enzyme-linked immunosorbent assay. The overall Toxocara seroprevalence among the study population (n = 2866) was 12.25%, and a significantly higher seroprevalence in psychiatric patients (16.40%, 73/445) than that in clinically healthy individuals (13.07%, 187/1431) and pregnant women (9.19%, 91/990) was revealed. Keeping dogs at home, contact with cats and dogs, and exposure with soil were found to be associated with Toxocara infection. Our findings indicate that human infection with Toxocara is common in eastern China, posing a significant public health concern.
PMCID: PMC4125144  PMID: 25101756
20.  MicroRNA in Alzheimer’s disease: an exploratory study in brain, cerebrospinal fluid and plasma 
MicroRNA (miRNA) may be potential biomarkers of Alzheimer’s disease (AD). The objective of this investigation was to demonstrate that miRNAs in human brain or biofluids are differentially expressed according to disease status, tissue type, neuritic plaque score or Braak stage. Post-mortem brain (PMB) miRNA were profiled using arrays and validated using quantitative RT-PCR (qRT-PCR). Five qRT-PCR-validated miRNAs were measured in an independent sample of PMB, cerebrospinal fluid and plasma from the same subjects. Plasma miR-15a was found to be associated with plaque score in the independent sample. In conclusion, miRNA present in human biofluids may offer utility as biomarkers of AD.
PMCID: PMC3967870  PMID: 23822153
Amyloid; biomarker; Braak stage; miR-15a; miR-370; miR-328; miR-138; miR 132; plaque
21.  Dipeptidyl‐Peptidase 4 Inhibition and the Vascular Effects of Glucagon‐like Peptide‐1 and Brain Natriuretic Peptide in the Human Forearm 
Dipeptidyl‐peptidase 4 (DPP4) inhibitors improve glycemic control in patients with diabetes mellitus by preventing the degradation of glucagon‐like peptide‐1 (GLP‐1). GLP‐1 causes vasodilation in animal models but also increases sympathetic activity; the effect of GLP‐1 in the human vasculature and how it is altered by DPP4 inhibition is not known. DPP4 also degrades the vasodilator brain natriuretic peptide (BNP) to a less potent metabolite. This study tested the hypothesis that DPP4 inhibition potentiates the vasodilator responses to GLP‐1 and BNP in the human forearm.
Method and Results
Seventeen healthy subjects participated in this randomized, double‐blinded, placebo‐controlled crossover study. On each study day, subjects received DPP4 inhibitor (sitagliptin 200 mg by mouth) or placebo. Sitagliptin increased forearm blood flow and decreased forearm vascular resistance without affecting mean arterial pressure and pulse. GLP‐1 and BNP were infused in incremental doses via brachial artery. Venous GLP‐1 concentrations were significantly higher during sitagliptin use, yet there was no effect of GLP‐1 on forearm blood flow in the presence or absence of sitagliptin. BNP caused dose‐dependent vasodilation; however, sitagliptin did not affect this response. GLP‐1 and BNP had no effect on net norepinephrine release.
These data suggest that GLP‐1 does not act as a direct vasodilator in humans and does not contribute to sympathetic activation. Sitagliptin does not regulate vascular function in healthy humans by affecting the degradation of GLP‐1 and BNP.
Clinical Trial Registration
URL: Unique identifier: NCT01413542.
PMCID: PMC4310400  PMID: 25158865
diabetes mellitus; dipeptidyl‐peptidase 4; glucagon‐like peptide‐1; natriuretic peptide; vasodilation
22.  Design and validation of a passive deposition sampler 
A new, passive particle deposition air sampler, called the Einstein–Lioy Deposition Sampler (ELDS), has been developed to fill a gap in passive sampling for near-field particle emissions. The sampler can be configured in several ways: with a protective hood for outdoor sampling, without a protective hood, and as a dust plate. In addition, there is an XRF-ready option that allows for direct sampling onto a filter-mounted XRF cartridge which can be used in conjunction with all configurations. A wind tunnel was designed and constructed to test the performance of different sampler configurations using a test dust with a known particle size distribution. The sampler configurations were also tested versus each other to evaluate whether or not the protective hood would affect the collected particle size distribution. A field study was conducted to test the sampler under actual environmental conditions and to evaluate its ability to collect samples for chemical analysis. Individual experiments for each configuration demonstrated precision of the sampler. The field experiment demonstrated the ability of the sampler to both collect mass and allow for the measurement of an environmental contaminant i.e. Cr6+. The ELDS was demonstrated to be statistically not different for Hooded and Non-Hooded models, compared to each other and the test dust; thus, it can be used indoors and outdoors in a variety of configurations to suit the user's needs.
PMCID: PMC4114154  PMID: 22820464
23.  Genotoxicity Study of Polysaccharide Fraction from Astragalus membranaceus's Aerial Parts 
Toxicological Research  2014;30(2):131-138.
Radix Astragali, the root of Astragalus (A.) membranaceus, has been applied in a variety of diseases for a long time in Asian countries such as Korea and China. In addition, the aerial parts such as leaves and stems of A. membranaceus have received a great deal of attention. Recently, the polysaccharide fraction showing a potent immunomoduating activity was isolated from the aerial parts of A. membranaceus. Thus, the aerial parts of A. membranaceus would be worthy enough for a food material and a dietary supplement. However, they should be safe even though valuable. In our previous study, it was estimated that NOAEL for female rats are 5000 mg/kg/day of the crude polysaccharide fraction from A. membranaceus-aboveground parts. As a series of safety evaluation, genotoxicity test for the crude polysaccharide fraction was carried out in this study. In conclusion, the three genotoxicity assays provided strong overall support that the crude polysaccharide fraction lacks mutagenic and/or clastogenic potential under the GLP-based test conditions. This indicates the aerial parts of A. membranaceus would be safe enough for a food material and a dietary supplement.
PMCID: PMC4112064  PMID: 25071923
Astragalus membranaceus; Aerial parts; Genotoxicity test; Polysaccharide
24.  The effect of various assisted hatching techniques on the mouse early embryo development 
In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers.
Four- to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed.
Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05).
Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.
PMCID: PMC4102692  PMID: 25045630
Assisted hatching; Cell number; Laser assisted hatching; Mechanical assisted hatching; Mouse embryo
25.  Is it possible to identify cases of coronary artery bypass graft postoperative surgical site infection accurately from claims data? 
Claims data has usually been used in recent studies to identify cases of healthcare-associated infection. However, several studies have indicated that the ICD-9-CM codes might be inappropriate for identifying such cases from claims data; therefore, several researchers developed alternative identification models to correctly identify more cases from claims data. The purpose of this study was to investigate three common approaches to develop alternative models for the identification of cases of coronary artery bypass graft (CABG) surgical site infection, and to compare the performance between these models and the ICD-9-CM model.
The 2005–2008 National Health Insurance claims data and healthcare-associated infection surveillance data from two medical centers were used in this study for model development and model verification. In addition to the use of ICD-9-CM codes, this study also used classification algorithms, a multivariable regression model, and a decision tree model in the development of alternative identification models. In the classification algorithms, we defined three levels (strict, moderate, and loose) of the criteria in terms of their strictness. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were used to evaluate the performance of each model.
The ICD-9-CM-based model showed good specificity and negative predictive value, but sensitivity and positive predictive value were poor. Performances of the other models were varied, except for negative predictive value. Among the models, the performance of the decision tree model was excellent, especially in terms of positive predictive value.
The accuracy of identification of cases of CABG surgical site infection is an important issue in claims data. Use of the decision tree model to identify such cases can improve the accuracy of patient-level outcome research. This model should be considered when performing future research using claims data.
PMCID: PMC4050397  PMID: 24884488
Administrative data; Identification model; CABG; Surgical site infection; Decision tree; Classification and regression tree

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