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1.  Engineered Holliday Junctions as Single-Molecule Reporters for Protein-DNA Interactions with Application to a MerR-family Regulator 
Journal of the American Chemical Society  2007;129(41):12461-12467.
Protein-DNA interactions are essential for gene maintenance, replication and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Since the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo- and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.
PMCID: PMC2528078  PMID: 17880214
2.  Clinicopathologic features and prognostic implications of NOK/STYK1 protein expression in non-small cell lung cancer 
BMC Cancer  2014;14:402.
The expression of novel oncogenic kinase (NOK), a member of the protein tyrosine kinase (PTK) family, has been observed in several human malignancies including non-small cell lung cancer (NSCLC). However, the clinic relevance of NOK expression in NSCLC remains unclear.
In this study, NOK expression in tumor cells was assessed using immunohistochemical methods in 191 patients with resected NSCLC. The association of NOK expression with clinicopathological parameters, including the Ki-67 labeling index (LI), was also evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of NOK expression on survival.
Data showed that NOK was expressed in 75.4% and 14.1% of cancer lesions and corresponding adjacent non-cancerous tissue, respectively. Out of all the clinicopathological factors analyzed, NOK expression was significantly correlated with the grade of tumor differentiation (P = 0.035), pTNM stage (P = 0.020), lymphatic metastasis (P = 0.005) and high Ki-67 LI (P < 0.001). NOK positive NSCLC patients had a significantly shorter survival time (P = 0.004, Log-rank test) and the prognostic significance of NOK expression was apparent in squamous cell carcinoma patients (P = 0.022). Multivariate analysis indicated that NOK expression may be an independent prognostic factor in NSCLC (hazard ratio [HR], 1.731; P = 0.043).
Our results indicate that NOK expression is of clinical significance and can serve as a prognostic biomarker in NSCLC.
PMCID: PMC4051150  PMID: 24894011
NOK; Oncogene; Lung cancer; NSCLC; Survival; Prognosis; Immunohistochemistry
3.  Pro-arrhythmic Effect of Blocking the Small Conductance Calcium Activated Potassium Channel in Isolated Canine Left Atrium 
Small conductance calcium activated potassium channels (SKCa) are voltage insensitive and are activated by intracellular calcium. Genome wide association studies revealed that a variant of SKca is associated with lone atrial fibrillation (AF) in humans. Roles of SKca in atrial arrhythmias remain unclear.
The purpose of this study was to determine roles of SKCa in atrial arrhythmias.
Optical mapping using isolated canine left atrium was performed. The optical action potential duration (APD) and induction of arrhythmia were evaluated before and after the addition of specific SKCa blockers, Apamin or UCL-1684.
SKCa blockade significantly increased APD80 (188±19 ms vs 147±11ms, p< 0.001). The pacing cycle length (PCL) thresholds to induce 2:2 alternans and wave breaks were prolonged by SKCa blockade. Increased APD heterogeneity was observed following SKCa blockade, as measured by the difference between maximum and minimum APD (39±4ms vs 26±5ms, p<0.05), by standard deviation (12.43±2.36ms vs 7.49±1.47ms, p<0.001), or by coefficient of variation (6.68±0.97% vs 4.90±0.84%, p<0.05). No arrhythmia was induced at baseline by S1–S2 protocol. After SKCa blockade, 4 out of 6 atria developed arrhythmia.
Blockade of SKCa promotes arrhythmia and prolongs the PCL threshold of 2:2 alternans and wave breaks in the canine left atrium. The proarrhythmic effect could be attributed to the increased APD heterogeneity in the canine left atrium. This study provides supportive evidence of GWAS studies showing association of KCNN3 and lone AF
PMCID: PMC3663880  PMID: 23376397
Atrial arrhythmia; SKCa; action potential duration; repolarization; optical mapping
4.  Low-Level Vagus Nerve Stimulation Upregulates Small Conductance Calcium Activated Potassium Channels in the Stellate Ganglion 
Small conductance calcium activated potassium (SK) channels are responsible for afterhyperpolarization that suppresses nerve discharges.
To test the hypotheses that low-level vagus nerve stimulation (LL-VNS) leads to the upregulation of SK2 proteins in the LSG.
Six dogs (Group 1) underwent 1-wk LL-VNS of the left cervical vagus nerve. Five normal dogs (Group 2) were used as control. SK2 protein levels were examined by western blotting. The ratio between SK2 and glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) levels was used as an arbitrary unit (AU).
We found higher SK2 expression in Group 1 (0.124 ± 0.049 AU) than Group 2 (0.085 ± 0.031 AU, P < 0.05). Immunostaining showed that the density of nerve structures stained with SK2 antibody was also higher in Group 1 (11,546 ± 7,271 μm2/mm2) than in Group 2 (5,321 ± 3,164 μm2/mm2, P < 0.05). There were significantly more ganglion cells without immunoreactivity to TH in Group 1 (11.4 ± 2.3%) than Group 2 (4.9 ± 0.7%; P < 0.05). The TH-negative ganglion cells mostly stained positive for choline acetyltransferase (ChAT) (95.9 ± 2.8% in Group 1 and 86.1 ± 4.4% in Group 2, P = 0.10). Immunofluorescence confocal microscopy revealed a significant decrease in the SK2 staining in the cytosol but an increase in the SK2 staining on the membrane of the ganglion cells in Group 1 compared to Group 2.
Left LL-VNS results in the upregulation of SK2 proteins, increased SK2 protein expression in the cell membrane and the increased TH-negative (mostly ChAT-positive) ganglion cells in the LSG. These changes may underlie the antiarrhythmic efficacy of LL-VNS in ambulatory dogs.
PMCID: PMC3671581  PMID: 23357541
Autonomic nervous system; Vagus nerve stimulation; Stellate ganglion; Small conductance calcium activated potassium channel; Western blot
5.  Insights into the Genetic Structure and Diversity of 38 South Asian Indians from Deep Whole-Genome Sequencing 
PLoS Genetics  2014;10(5):e1004377.
South Asia possesses a significant amount of genetic diversity due to considerable intergroup differences in culture and language. There have been numerous reports on the genetic structure of Asian Indians, although these have mostly relied on genotyping microarrays or targeted sequencing of the mitochondria and Y chromosomes. Asian Indians in Singapore are primarily descendants of immigrants from Dravidian-language–speaking states in south India, and 38 individuals from the general population underwent deep whole-genome sequencing with a target coverage of 30X as part of the Singapore Sequencing Indian Project (SSIP). The genetic structure and diversity of these samples were compared against samples from the Singapore Sequencing Malay Project and populations in Phase 1 of the 1,000 Genomes Project (1 KGP). SSIP samples exhibited greater intra-population genetic diversity and possessed higher heterozygous-to-homozygous genotype ratio than other Asian populations. When compared against a panel of well-defined Asian Indians, the genetic makeup of the SSIP samples was closely related to South Indians. However, even though the SSIP samples clustered distinctly from the Europeans in the global population structure analysis with autosomal SNPs, eight samples were assigned to mitochondrial haplogroups that were predominantly present in Europeans and possessed higher European admixture than the remaining samples. An analysis of the relative relatedness between SSIP with two archaic hominins (Denisovan, Neanderthal) identified higher ancient admixture in East Asian populations than in SSIP. The data resource for these samples is publicly available and is expected to serve as a valuable complement to the South Asian samples in Phase 3 of 1 KGP.
Author Summary
Indians of South Asia has long been a population of interest to a wide audience, due to its unique diversity. We have deep-sequenced 38 individuals of Indian descent residing in Singapore (SSIP) in an effort to illustrate their diversity from a whole-genome standpoint. Indeed, among Asians in our population panel, SSIP was most diverse, followed by the Malays in Singapore (SSMP). Their diversity is further observed in the population's chromosome Y haplogroup and mitochondria haplogroup profiles; individuals with European-dominant haplogroups had greater proportion of European admixture. Among variants (single nucleotide polymorphism and small insertions/deletions) discovered in SSIP, 21.69% were novel with respect to previous sequencing projects. In addition, some 14 loss-of-function variants (LOFs) were associated to cancer, Type II diabetes, and cholesterol levels. Finally, D statistic test with ancient hominids concurred that there was gene flow to East Asians compared to South Asians.
PMCID: PMC4022468  PMID: 24832686
6.  Apamin Does Not Inhibit Human Cardiac Na+ Current, L-type Ca2+ Current or Other Major K+ Currents 
PLoS ONE  2014;9(5):e96691.
Apamin is commonly used as a small-conductance Ca2+-activated K+ (SK) current inhibitor. However, the specificity of apamin in cardiac tissues remains unclear.
To test the hypothesis that apamin does not inhibit any major cardiac ion currents.
We studied human embryonic kidney (HEK) 293 cells that expressed human voltage-gated Na+, K+ and Ca2+ currents and isolated rabbit ventricular myocytes. Whole-cell patch clamp techniques were used to determine ionic current densities before and after apamin administration.
Ca2+ currents (CACNA1c+CACNB2b) were not affected by apamin (500 nM) (data are presented as median [25th percentile;75th percentile] (from –16 [–20;–10] to –17 [–19;–13] pA/pF, P = NS), but were reduced by nifedipine to –1.6 [–3.2;–1.3] pA/pF (p = 0.008). Na+ currents (SCN5A) were not affected by apamin (from –261 [–282;–145] to –268 [–379;–132] pA/pF, P = NS), but were reduced by flecainide to –57 [–70;–47] pA/pF (p = 0.018). None of the major K+ currents (IKs, IKr, IK1 and Ito) were inhibited by 500 nM of apamin (KCNQ1+KCNE1, from 28 [20]; [37] to 23 [18]; [32] pA/pF; KCNH2+KCNE2, from 28 [24]; [30] to 27 [24]; [29] pA/pF; KCNJ2, from –46 [–48;–40] to –46 [–51;–35] pA/pF; KCND3, from 608 [505;748] to 606 [454;684]). Apamin did not inhibit the INa or ICaL in isolated rabbit ventricular myocytes (INa, from –67 [–75;–59] to –68 [–71;–59] pA/pF; ICaL, from –16 [–17;–14] to –14 [–15;–13] pA/pF, P = NS for both).
Apamin does not inhibit human cardiac Na+ currents, L-type Ca2+ currents or other major K+ currents. These findings indicate that apamin is a specific SK current inhibitor in hearts as well as in other organs.
PMCID: PMC4010514  PMID: 24798465
7.  Host-Microbiome Interactions in Alcoholic Liver Disease 
Gut and Liver  2014;8(3):237-241.
Alcoholic liver disease is a leading cause of morbidity and liver-related death worldwide. Intestinal bacterial overgrowth and dysbiosis induced by ethanol ingestion play an important role in the pathogenesis of alcoholic liver disease. After exposure to alcohol in the lumen, enteric bacteria alter their metabolism and thereby disturb intestinal homeostasis. Disruption of the mucosal barrier results in the translocation of microbial products that contribute to liver disease by inducing hepatic inflammation. In this review, we will discuss the effects of alcohol on the intestinal microbiome, and in particular, its effects on bacterial metabolism, bacterial translocation and ecological balance. A better understanding of the interactions among alcohol, the host and the microbiome will reveal new targets for therapy and lead to new treatments.
PMCID: PMC4026639  PMID: 24827618
Alcoholic liver injury; Microbiota; Permeability; Metabolism
8.  Electroanatomic Remodeling of the Left Stellate Ganglion After Myocardial Infarction 
The purpose of this study was to evaluate the changes of left stellate ganglionic nerve activity (SGNA) and left thoracic vagal nerve activity (VNA) after acute myocardial infarction (MI).
Whether MI results in remodeling of extracardiac nerve activity remains unclear.
We implanted radiotransmitters to record the SGNA, VNA, and electrocardiogram in 9 ambulatory dogs. After baseline monitoring, MI was created by 1-h balloon occlusion of the coronary arteries. The dogs were then continuously monitored for 2 months. Both stellate ganglia were stained for growth-associated protein 43 and synaptophysin. The stellate ganglia from 5 normal dogs were used as control.
MI increased 24-h integrated SGNA from 7.44 ± 7.19 Ln(Vs)/day at baseline to 8.09 ± 7.75 Ln(Vs)/day after the MI (p < 0.05). The 24-h integrated VNA before and after the MI was 5.29 ± 5.04 Ln(Vs)/day and 5.58 ± 5.15 Ln(Vs)/day, respectively (p < 0.05). A significant 24-h circadian variation was noted for the SGNA (p < 0.05) but not the VNA. The SGNA/VNA ratio also showed significant circadian variation. The nerve densities from the left SG were 63,218 ± 34,719 μm2/mm2 and 20,623 ± 4,926 μm2/mm2 for growth-associated protein 43 (p < 0.05) and were 32,116 ± 8,190 μm2/mm2 and 16,326 ± 4,679 μm2/mm2 for synaptophysin (p < 0.05) in MI and control groups, respectively. The right SG also showed increased nerve density after MI (p < 0.05).
MI results in persistent increase in the synaptic density of bilateral stellate ganglia and is associated with increased SGNA and VNA. There is a circadian variation of the SGNA/VNA ratio. These data indicate significant remodeling of the extracardiac autonomic nerve activity and structures after MI.
PMCID: PMC3975658  PMID: 22381432
acute myocardial infarction; autonomic nervous system; nerve recordings
9.  Apamin Sensitive Potassium Current Modulates Action Potential Duration Restitution and Arrhythmogenesis of Failing Rabbit Ventricles 
Apamin-sensitive K currents (IKAS) are upregulated in heart failure (HF). We hypothesize that apamin can flatten action potential duration restitution (APDR) curve and reduce ventricular fibrillation (VF) duration in failing ventricles.
Methods and Results
We simultaneously mapped membrane potential and intracellular Ca (Cai) in 7 rabbits hearts with pacing-induced HF and in 7 normal hearts. A dynamic pacing protocol was used to determine APDR at baseline and after apamin (100 nM) infusion. Apamin did not change APD80 in normal ventricles, but prolonged APD80 in failing ventricles at either long (≥300 ms) or short (≤170 ms) pacing cycle length (PCL), but not at intermediate PCL. The maximal slope of APDR curve was 2.03 [95% CI, 1.73 to 2.32] in failing ventricles and 1.26 [95% CI, 1.13 to 1.40] in normal ventricles at baseline (p=0.002). After apamin administration, the maximal slope of APDR in failing ventricles decreased to 1.43 [95% CI, 1.01 to 1.84] (p=0.018) whereas no significant changes were observed in normal ventricles. During VF in failing ventricles, the number of phase singularities (baseline vs apamin, 4.0 vs 2.5), dominant frequency (13.0 Hz vs 10.0 Hz), and VF duration (160 s vs 80 s) were all significantly (p<0.05) decreased by apamin.
Apamin prolongs APD at long and short, but not at intermediate PCL in failing ventricles. IKAS upregulation may be antiarrhythmic by preserving the repolarization reserve at slow heart rate, but is proarrhythmic by steepening the slope of APDR curve which promotes the generation and maintenance of VF.
PMCID: PMC3678988  PMID: 23420832
ventricular fibrillation; optical mapping; experimental models heart failure; electrophysiology
10.  Sympathetic nerve fibers and ganglia in canine cervical vagus nerves: Localization and quantitation 
Cervical vagal nerve (CVN) stimulation may improve left ventricular ejection fraction in patients with heart failure.
To test the hypothesis that sympathetic structures are present in the CVN and to describe the location and quantitate these sympathetic components of the CVN.
We performed immunohistochemical studies of the CVN from 11 normal dogs and simultaneously recorded stellate ganglion nerve activity, left thoracic vagal nerve activity, and subcutaneous electrocardiogram in 2 additional dogs.
A total of 28 individual nerve bundles were present in the CVNs of the first 11 dogs, with an average of 1.87 ± 1.06 per dog. All CVNs contain tyrosine hydroxylase-positive (sympathetic) nerves, with a total cross-sectional area of 0.97 ± 0.38 mm2. The sympathetic nerves were nonmyelinated, typically located at the periphery of the nerve bundles and occupied 0.03%–2.80% of the CVN cross-sectional area. Cholineacetyltransferase-positive nerve fibers occupied 12.90%–42.86% of the CVN cross-sectional areas. Ten of 11 CVNs showed tyrosine hydroxylase and cholineacetyltransferase colocalization. In 2 dogs with nerve recordings, we documented heart rate acceleration during spontaneous vagal nerve activity in the absence of stellate ganglion nerve activity.
Sympathetic nerve fibers are invariably present in the CVNs of normal dogs and occupy in average up to 2.8% of the cross-sectional area. Because sympathetic nerve fibers are present in the periphery of the CVNs, they may be susceptible to activation by electrical stimulation. Spontaneous activation of the sympathetic component of the vagal nerve may accelerate the heart rate.
PMCID: PMC3758134  PMID: 23246597
Cervical vagus nerves; Sympathetic nerves; Ganglion cells; Heart failure; Vagal nerve stimulation
11.  Characterization of Maltocin P28, a Novel Phage Tail-Like Bacteriocin from Stenotrophomonas maltophilia 
Applied and Environmental Microbiology  2013;79(18):5593-5600.
Stenotrophomonas maltophilia is an important global opportunistic pathogen for which limited therapeutics are available because of the emergence of multidrug-resistant strains. A novel bacteriocin, maltocin P28, which is produced by S. maltophilia strain P28, may be the first identified phage tail-like bacteriocin from S. maltophilia. Maltocin P28 resembles a contractile but nonflexible phage tail structure based on electron microscopy, and it is sensitive to trypsin, proteinase K, and heat. SDS-PAGE analysis of maltocin P28 revealed two major protein bands of approximately 43 and 20 kDa. The N-terminal amino acid residues of these two major subunits were sequenced, and the maltocin P28 gene cluster was located on the S. maltophilia P28 chromosome. Our sequence analysis results indicate that this maltocin gene cluster consists of 23 open reading frames (ORFs), and that its gene organization is similar to that of the P2 phage genome and R2 pyocin gene cluster. ORF17 and ORF18 encode the two major structural proteins, which correspond to gpFI (tail sheath) and gpFII (tail tube) of P2 phage, respectively. We found that maltocin P28 had bactericidal activity against 38 of 81 tested S. maltophilia strains. Therefore, maltocin P28 is a promising therapeutic substitute for antibiotics for S. maltophilia infections.
PMCID: PMC3754179  PMID: 23835182
12.  Draft Genome Sequence of Bacillus subtilis Type Strain B7-S, Which Converts Ferulic Acid to Vanillin 
Genome Announcements  2014;2(1):e00025-14.
The Bacillus subtilis type strain B7-S was obtained through induction with ferulic acid. Here, we present the draft genome of strain B7-S, which contains 5,313,924 bp, with a G+C content of 35.8%, 5,135 protein-coding genes, and 40 tRNA-encoding genes.
PMCID: PMC3924361  PMID: 24526629
13.  Association of Cholecystectomy with Metabolic Syndrome in a Chinese Population 
PLoS ONE  2014;9(2):e88189.
An association between cholecystectomy and metabolic syndrome has not been fully established. Here we analyzed the association between cholecystectomy and metabolic syndrome in a Chinese population of 5672 subjects who undergone annual health checkups at the First Affiliated Hospital, College of Medicine, Zhejiang University between January 2011 and December 2012. The prevalences of gallstones, cholecystectomy and metabolic syndrome were 6.0%, 3.6%, and 32.5%, respectively. The prevalence of metabolic syndrome was significantly higher in subjects with a history of cholecystectomy (63.5%) than in those with gallstones (47.0%) or in those without gallstone disease (30.3%; P<0.01 for both). Multivariate logistic regression analysis showed that cholecystectomy was significantly associated with increased risk of metabolic syndrome (OR = 1.872; 95% CI: 1.193–2.937). However, the association of gallstones with metabolic syndrome was not statistically significant (OR = 1.267; 95% CI: 0.901–1.782). Altogether, our results suggest that cholecystectomy significantly increases the risk of metabolic syndrome.
PMCID: PMC3914934  PMID: 24505425
14.  A Ser252Trp Mutation in Fibroblast Growth Factor Receptor 2 (FGFR2) Mimicking Human Apert Syndrome Reveals an Essential Role for FGF Signaling in the Regulation of Endochondral Bone Formation 
PLoS ONE  2014;9(1):e87311.
A S252W mutation of fibroblast growth factor receptor 2 (FGFR2), which is responsible for nearly two-thirds of Apert syndrome (AS) cases, causes retarded development of the skeleton and skull malformation resulting from premature fusion of the craniofacial sutures. We utilized a Fgfr2+/S252W mouse (a knock-in mouse model mimicking human AS) to demonstrate decreased bone mass due to reduced trabecular bone volume, reduced bone mineral density, and shortened growth plates in the long bones. In vitro bone mesenchymal stem cells (BMSCs) culture studies revealed that the mutant mice showed reduced BMSC proliferation, a reduction in chondrogenic differentiation, and reduced mineralization. Our results suggest that these phenomena are caused by up-regulation of p38 and Erk1/2 phosphorylation. Treatment of cultured mutant bone rudiments with SB203580 or PD98059 resulted in partial rescue of the bone growth retardation. The p38 signaling pathway especially was found to be responsible for the retarded long bone development. Our data indicate that the S252W mutation in FGFR2 directly affects endochondral ossification, resulting in growth retardation of the long bone. We also show that the p38 and Erk1/2 signaling pathways partially mediate the effects of the S252W mutation of FGFR2 on long bone development.
PMCID: PMC3904987  PMID: 24489893
15.  Growth Promotion-Related miRNAs in Oncidium Orchid Roots Colonized by the Endophytic Fungus Piriformospora indica 
PLoS ONE  2014;9(1):e84920.
Piriformospora indica, an endophytic fungus of Sebacinales, colonizes the roots of a wide range of host plants and establishes various benefits for the plants. In this work, we describe miRNAs which are upregulated in Oncidium orchid roots after colonization by the fungus. Growth promotion and vigorous root development were observed in Oncidium hybrid orchid, while seedlings were colonized by P. indica. We performed a genome-wide expression profiling of small RNAs in Oncidium orchid roots either colonized or not-colonized by P. indica. After sequencing, 24,570,250 and 24744,141 clean reads were obtained from two libraries. 13,736 from 17,036,953 unique sequences showed homology to either 86 miRNA families described in 41 plant species, or to 46 potential novel miRNAs, or to 51 corresponding miRNA precursors. The predicted target genes of these miRNAs are mainly involved in auxin signal perception and transduction, transcription, development and plant defense. The expression analysis of miRNAs and target genes demonstrated the regulatory functions they may participate in. This study revealed that growth stimulation of the Oncidium orchid after colonization by P. indica includes an intricate network of miRNAs and their targets. The symbiotic function of P. indica on Oncidium orchid resembles previous findings on Chinese cabbage. This is the first study on growth regulation and development of Oncidium orchid by miRNAs induced by the symbiotic fungus P. indica.
PMCID: PMC3883679  PMID: 24409313
16.  Carvedilol Analogue Inhibits Triggered Activities Evoked by Both Early and Delayed Afterdepolarizations 
Carvedilol and its analogues suppress delayed afterdepolarizations (DADs) and catecholaminergic polymorphic ventricular tachycardias by direct action on the cardiac ryanodine receptor (RyR2).
We tested a hypothesis that carvedilol analogue may also prevent triggered activities (TAs) through the suppression of early afterdepolarizations (EADs).
Intracellular Ca2+ and membrane voltage were simultaneously recorded using optical mapping technique in Langendorff-perfused mouse and rabbit hearts to study the effect of carvedilol analogue, VK-II-36 that does not have significant beta-blocking effects.
Spontaneous intracellular Ca2+ elevations (SCaEs) during diastole was induced by rapid ventricular pacing and isoproterenol infusion in intact rabbit ventricles. Systolic and diastolic SCaEs were simultaneously noted in Langendorff-perfused RyR2 R4496+/− mouse hearts after creating atrioventricular block. VK-II-36 effectively suppressed SCaEs and eliminated TAs observed in both mouse and rabbit ventricles. We tested the effect of VK-II-36 on EADs using a rabbit model of acquired long QT syndrome in which phase-2 and phase-3 EADs were observed in association with systolic SCaEs. VK-II-36 abolished the systolic SCaEs and phase-2 EADs, and greatly decreased the dispersion of repolarization and the amplitude of phase-3 EADs. VK-II-36 completely prevented EAD-mediated TAs in all ventricles studied.
A carvedilol analogue, VK-II-36 inhibits ventricular tachyarrhythmias in intact mouse and rabbit ventricles by suppression of SCaEs, independent of beta-blocking activity. The RyR2 may be a potential target for treating focal ventricular arrhythmias triggered by either EADs or DADs.
PMCID: PMC3534812  PMID: 22982970
afterdepolarization; intracellular calcium; long-QT syndrome
17.  Changes of phasic and tonic smooth muscle function of jejunum in type 2 diabetic Goto-Kakizaki rats 
World Journal of Diabetes  2013;4(6):339-348.
AIM: To generate phasic and tonic stress-strain curves for evaluation of intestinal smooth muscle function in type 2 diabetic rats during active and passive conditions.
METHODS: Seven diabetic Goto-Kakizaki (GK) male rats, 32-wk old (GK group), and 9 age-matched normal Wistar rats (Normal group) were included in the study. Jejunal segments were distended up to a pressure of 10 cm H2O in an organ bath containing 37 °C Krebs solution with addition of carbachol (CA). The pressure and outer diameter changes were synchronously recorded. Passive conditions were obtained using calcium-free Krebs solution containing ethylene glycol tetraacetic acid and papaverine. Total phasic, tonic and passive circumferential stress and strain were computed from the diameter and pressure data with reference to the zero-stress state geometry. The active phasic and tonic stresses were defined as the total phasic and tonic stresses minus the passive stress.
RESULTS: Diabetes increased jejunal mucosa and muscle layer thicknesses compared to the Normal group (mucosa, 755.8 ± 63.3 vs 633.1 ± 59.1 μm, P < 0.01; muscle, 106.3 ± 12.9 vs 85.2 ± 11.7 μm, P < 0.05). The pressure and stress thresholds were decreased in the GK group after CA application compared to distensions without CA application (pressure, 1.01 ± 0.07 vs 1.99 ± 0.19 cmH2O, P < 0.01; stress, 0.11 ± 0.01 vs 0.24 ± 0.02 kPa, P < 0.01). CA application did not change the pressure and stress threshold in the Normal group (pressure, 2.13 ± 0.32 vs 2.34 ± 0.32 cm H2O, P > 0.05; stress, 0.25 ± 0.03 vs 0.35 ± 0.06 kPa, P > 0.05). The amplitude of total phasic, total tonic, active phasic and active tonic circumferential stresses did not differ for the distensions without CA application between the GK group and the Normal group. However, the total phasic and total tonic stresses increased after CA application in the GK group compared those in the Normal group. When normalized to muscle layer thickness, the amplitude of active stresses before CA application was lowest in the GK group compared with the Normal group. No difference was found during CA application.
CONCLUSION: The stress generated by intestinal muscle normalized to the muscle layer thickness was lowest in GK rats compared to normal rats whereas the response to CA stimulation was preserved.
PMCID: PMC3874494  PMID: 24379925
Intestine; Diabetes; Muscle function; Stress-strain curves; Carbachol; Rat
18.  Physiological Mechanisms for High Salt Tolerance in Wild Soybean (Glycine soja) from Yellow River Delta, China: Photosynthesis, Osmotic Regulation, Ion Flux and antioxidant Capacity 
PLoS ONE  2013;8(12):e83227.
Glycine soja (BB52) is a wild soybean cultivar grown in coastal saline land in Yellow River Delta, China. In order to reveal the physiological mechanisms adapting to salinity, we examined photosynthesis, ion flux, antioxidant system and water status in Glycine soja under NaCl treatments, taking a cultivated soybean, ZH13, as control. Upon NaCl exposure, higher relative water content and water potential were maintained in the leaf of BB52 than ZH13, which might depend on the more accumulation of osmotic substances such as glycinebetaine and proline. Compared with ZH13, activities of antioxidant enzymes including superoxide dismutase, catalase, ascorbate peroxidase and contents of ascorbate, glutathione and phenolics were enhanced to a higher level in BB52 leaf under NaCl stress, which could mitigate the salt-induced oxidative damage in BB52. Consistently, lipid peroxidation indicated by malondialdehyde content was lower in BB52 leaf. Photosynthetic rate (Pn) was decreased by NaCl stress in BB52 and ZH13, and the decrease was greater in ZH13. The decreased Pn in BB52 was mainly due to stomatal limitation. The inhibited activation of rubisco enzyme in ZH13 due to the decrease of rubisco activase content became an important limiting factor of Pn, when NaCl concentration increased to 200 mM. Rubisco activase in BB52 was not affected by NaCl stress. Less negative impact in BB52 derived from lower contents of Na+ and Cl- in the tissues, and non-invasive micro-test technique revealed that BB52 roots had higher ability to extrude Na+ and Cl-. Wild soybean is a valuable genetic resource, and our study may provide a reference for molecular biologist to improve the salt tolerance of cultivated soybean in face of farmland salinity.
PMCID: PMC3861505  PMID: 24349468
19.  Intracellular Calcium Dynamics, Shortened Action Potential Duration and Late-phase 3 Early Afterdepolarization in Langendorff-Perfused Rabbit Ventricles 
To elucidate the mechanism of late-phase 3 early after depolarization (EAD) in ventricular arrhythmogenesis, we hypothesized that intracellular calcium (Cai) overloading and action potential duration (APD) shortening may promote late phase 3 EAD and triggered activity, leading to development of ventricular fibrillation (VF).
Methods and Results
In isolated rabbit hearts, we performed microelectrode recording and simultaneous dual optical mapping of transmembrane potential (Vm) and Cai transient on left ventricular endocardium. An IKATP channel opener, pinacidil, was used to abbreviate action potential duration (APD). Rapid-pacing was then performed. Upon abrupt cessation of rapid pacing with cycle lengths of 60–200 ms, there were APD90 prolongation and the corresponding Cai overloading in the first post-pacing beats. The duration of Cai transient recovered to 50% (DCaT50) and 90% (DCaT90) in the first post-pacing beats was significantly longer than baseline. Abnormal Cai elevation coupled with shortened APD produced late-phase 3 EAD induced triggered activity and VF. In additional 6 preparations, the heart tissues were treated with BAPTA-AM, a calcium chelator. BAPTA-AM significantly reduced the maximal Cai amplitude (26.4±3.5% of the control; p<0.001) and the duration of Cai transients in the mapped region, preventing the development of EAD and triggered activity that initiated VF.
IKATP channel activation along with Cai overloading are associated with the development of late phase 3 EAD and VF. Because acute myocardial ischemia activates the IKATP channel, late phase 3 EADs may be a mechanism for VF initiation during acute myocardial ischemia.
PMCID: PMC3479328  PMID: 22809087
ventricular fibrillation; early afterdepolarization; triggered activity; calcium; APD shortening
20.  Pathways-Driven Sparse Regression Identifies Pathways and Genes Associated with High-Density Lipoprotein Cholesterol in Two Asian Cohorts 
PLoS Genetics  2013;9(11):e1003939.
Standard approaches to data analysis in genome-wide association studies (GWAS) ignore any potential functional relationships between gene variants. In contrast gene pathways analysis uses prior information on functional structure within the genome to identify pathways associated with a trait of interest. In a second step, important single nucleotide polymorphisms (SNPs) or genes may be identified within associated pathways. The pathways approach is motivated by the fact that genes do not act alone, but instead have effects that are likely to be mediated through their interaction in gene pathways. Where this is the case, pathways approaches may reveal aspects of a trait's genetic architecture that would otherwise be missed when considering SNPs in isolation. Most pathways methods begin by testing SNPs one at a time, and so fail to capitalise on the potential advantages inherent in a multi-SNP, joint modelling approach. Here, we describe a dual-level, sparse regression model for the simultaneous identification of pathways and genes associated with a quantitative trait. Our method takes account of various factors specific to the joint modelling of pathways with genome-wide data, including widespread correlation between genetic predictors, and the fact that variants may overlap multiple pathways. We use a resampling strategy that exploits finite sample variability to provide robust rankings for pathways and genes. We test our method through simulation, and use it to perform pathways-driven gene selection in a search for pathways and genes associated with variation in serum high-density lipoprotein cholesterol levels in two separate GWAS cohorts of Asian adults. By comparing results from both cohorts we identify a number of candidate pathways including those associated with cardiomyopathy, and T cell receptor and PPAR signalling. Highlighted genes include those associated with the L-type calcium channel, adenylate cyclase, integrin, laminin, MAPK signalling and immune function.
Author Summary
Genes do not act in isolation, but interact in complex networks or pathways. By accounting for such interactions, pathways analysis methods hope to identify aspects of a disease or trait's genetic architecture that might be missed using more conventional approaches. Most existing pathways methods take a univariate approach, in which each variant within a pathway is separately tested for association with the phenotype of interest. These statistics are then combined to assess pathway significance. As a second step, further analysis can reveal important genetic variants within significant pathways. We have previously shown that a joint-modelling approach using a sparse regression model can increase the power to detect pathways influencing a quantitative trait. Here we extend this approach, and describe a method that is able to simultaneously identify pathways and genes that may be driving pathway selection. We test our method using simulations, and apply it to a study searching for pathways and genes associated with high-density lipoprotein cholesterol in two separate East Asian cohorts.
PMCID: PMC3836716  PMID: 24278029
21.  Helicobacter pylori Infection Is Associated with the Presence of Thyroid Nodules in the Euthyroid Population 
PLoS ONE  2013;8(11):e80042.
Helicobacter pylori infection is associated with extragastric diseases. The thyroid may be one of the targets of chronic inflammation. Here, we sought to investigate whether H. pylori infections were associated with the presence of thyroid nodules. A total of 988 euthyroid subjects from China were included in this cross-sectional study. Four hundred thirty-five (44.0%) subjects were diagnosed as having thyroid nodules, and 486 (49.2%) were diagnosed with H. pylori infections. The thyroid nodules group had a higher proportion of H. pylori infections than the control group (P = 0.002). Free thyroxine (FT4) levels were lower and the prevalence of thyroid nodules was higher in patients with H. pylori infection compared to those without infection, even after adjustment for age, gender, and body mass index (BMI; all P < 0.05). The prevalence of H. pylori infection showed a decreasing trend as serum FT4 level increased (Ptrend = 0.020). Stepwise logistic regression analysis showed that H. pylori infection was significantly associated with the risk of thyroid nodules (odds ratio: 1.390, 95% confidence interval: 1.059–1.824, P = 0.018). Our results suggested that H. pylori infections were positively associated with the presence of thyroid nodules in the euthyroid population, whose thyroid functions were in the reference range.
PMCID: PMC3823768  PMID: 24244604
22.  A Study Assessing the Association of Glycated Hemoglobin A1C (HbA1C) Associated Variants with HbA1C, Chronic Kidney Disease and Diabetic Retinopathy in Populations of Asian Ancestry 
PLoS ONE  2013;8(11):e79767.
Glycated hemoglobin A1C (HbA1C) level is used as a diagnostic marker for diabetes mellitus and a predictor of diabetes associated complications. Genome-wide association studies have identified genetic variants associated with HbA1C level. Most of these studies have been conducted in populations of European ancestry. Here we report the findings from a meta-analysis of genome-wide association studies of HbA1C levels in 6,682 non-diabetic subjects of Chinese, Malay and South Asian ancestries. We also sought to examine the associations between HbA1C associated SNPs and microvascular complications associated with diabetes mellitus, namely chronic kidney disease and retinopathy. A cluster of 6 SNPs on chromosome 17 showed an association with HbA1C which achieved genome-wide significance in the Malays but not in Chinese and Asian Indians. No other variants achieved genome-wide significance in the individual studies or in the meta-analysis. When we investigated the reproducibility of the findings that emerged from the European studies, six loci out of fifteen were found to be associated with HbA1C with effect sizes similar to those reported in the populations of European ancestry and P-value ≤ 0.05. No convincing associations with chronic kidney disease and retinopathy were identified in this study.
PMCID: PMC3820602  PMID: 24244560
23.  Association between Tumor Necrosis Factor-α 308G/A Gene Polymorphism and Silicosis Susceptibility: A Meta-Analysis 
PLoS ONE  2013;8(10):e76614.
Tumor necrosis factor-α (TNF-α) 308 G/A gene polymorphism has been reported to be associated with susceptibility to silicosis. However, the relevant study results are still inconsistent.
Objective and Methods
A meta-analysis was performed in order to drive a more precise estimation of the relationship between TNF-α-308 G/A gene polymorphism and susceptibility to silicosis. Electronic databases were searched and nine separate studies were included. The pooled odds ratios (ORs) and the corresponding 95% confidence internal (CI) were calculated by a fixed effect model.
A total of 1267 cases and 1214 controls were included. In the overall analysis, significantly increased silicosis risk was found (for GA+AA vs. GG OR=1.45, 95%CI: 1.20-1.760, P=1.58E4; for GA vs. GG: OR=1.53, 95%CI=1.25-1.86, P=3.11E5; for A allele vs. G allele: OR=1.27, 95%CI=1.08-1.50, P= 0.004). In the subgroup analysis, significantly increased silicosis risk was also found among Asians (for GA+AA vs. GG: OR=1.63, 95%CI=1.27-2.08, P=1.01E4), for GA vs. GG: OR=1.71, 95%CI=1.33-2.20, P=3.44E5), for A allele vs. G allele: OR=1.45, 95%CI=1.17-1.80, P=0.001). However, no significantly increased risk was found among non-Asians for all genetic models.
TNF-α-308 G/A polymorphism might lead to an increased risk of silicosis susceptibility, especially for Asians. However, further studies with large sample sizes should be conducted to confirm the association.
PMCID: PMC3790741  PMID: 24124578
24.  Mineral-Coated Polymer Membranes with Superhydrophilicity and Underwater Superoleophobicity for Effective Oil/Water Separation 
Scientific Reports  2013;3:2776.
Oil-polluted water is a worldwide problem due to the increasing industrial oily wastewater and the frequent oil spill accidents. Here, we report a novel kind of superhydrophilic hybrid membranes for effective oil/water separation. They were prepared by depositing CaCO3-based mineral coating on PAA-grafted polypropylene microfiltration membranes. The rigid mineral-coating traps abundant water in aqueous environment and forms a robust hydrated layer on the membrane pore surface, thus endowing the membranes with underwater superoleophobicity. Under the drive of either gravity or external pressure, the hybrid membranes separate a range of oil/water mixtures effectively with high water flux (>2000 L m−2 h−1), perfect oil/water separation efficiency (>99%), high oil breakthrough pressure (>140 kPa) and low oil fouling. The oil/water mixtures include not only free mixtures but also oil-in-water emulsions. Therefore, the mineral-coated membrane enables an efficient and energy-saving separation for various oil/water mixtures, showing attractive potential for practical oil/water separation.
PMCID: PMC3784956  PMID: 24072204
25.  The maturation zone is an important target of Piriformospora indica in Chinese cabbage roots 
Journal of Experimental Botany  2013;64(14):4529-4540.
The mutualistic symbiont Piriformospora indica exhibits a great potential in agriculture. The interaction between P. indica and Chinese cabbage (Brassica campestris cv. Chinensis) results in growth and biomass promotion of the host plant and in particular in root hair development. The resulting highly bushy root phenotype of colonized Chinese cabbage seedlings differs substantially from reports of other plant species, which prompted the more detailed study of this symbiosis. A large-scale expressed sequence tag (EST) data set was obtained from a double-subtractive EST library, by subtracting the cDNAs of Chinese cabbage root tissue and of P. indica mycelium from those of P. indica-colonized root tissue. The analysis revealed ~700 unique genes rooted in 141 clusters and 559 singles. A total of 66% of the sequences could be annotated in the NCBI GenBank. Genes which are stimulated by P. indica are involved in various types of transport, carbohydrate metabolism, auxin signalling, cell wall metabolism, and root development, including the root hair-forming phosphoinositide phosphatase 4. For 20 key genes, induction by fungal colonization was confirmed kinetically during the interaction by real-time reverse transcription–PCR. Moreover, the auxin concentration increases transiently after exposure of the roots to P. indica. Microscopic analyses demonstrated that the development of the root maturation zone is the major target of P. indica in Chinese cabbage. Taken together, the symbiotic interaction between Chinese cabbage and P. indica is a novel model to study root growth promotion which, in turn, is important for agriculture and plant biotechnology.
PMCID: PMC3808330  PMID: 24006423
Chinese cabbage; growth and biomass promotion; mutualistic symbiosis; Piriformospora indica; root maturation zone; subtractive EST library

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