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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.  Early afterdepolarizations in cardiac myocytes: beyond reduced repolarization reserve 
Cardiovascular Research  2013;99(1):6-15.
Early afterdepolarizations (EADs) are secondary voltage depolarizations during the repolarizing phase of the action potential, which can cause lethal cardiac arrhythmias. The occurrence of EADs requires a reduction in outward current and/or an increase in inward current, a condition called reduced repolarization reserve. However, this generalized condition is not sufficient for EAD genesis and does not explain the voltage oscillations manifesting as EADs. Here, we summarize recent progress that uses dynamical theory to build on and advance our understanding of EADs beyond the concept of repolarization reserve, towards the goal of developing a holistic and integrative view of EADs and their role in arrhythmogenesis. We first introduce concepts from nonlinear dynamics that are relevant to EADs, namely, Hopf bifurcation leading to oscillations and basin of attraction of an equilibrium or oscillatory state. We then present a theory of phase-2 EADs in nonlinear dynamics, which includes the formation of quasi-equilibrium states at the plateau voltage, their stabilities, and the bifurcations leading to and terminating the oscillations. This theory shows that the L-type calcium channel plays a unique role in causing the nonlinear dynamical behaviours necessary for EADs. We also summarize different mechanisms of phase-3 EADs. Based on the dynamical theory, we discuss the roles of each of the major ionic currents in the genesis of EADs, and potential therapeutic targets.
PMCID: PMC3687754  PMID: 23619423
Early afterdepolarizations; Repolarization reserve; Nonlinear dynamics; Oscillation; Arrhythmias
3.  Deficiency of intestinal mucin-2 ameliorates experimental alcoholic liver disease in mice 
Hepatology (Baltimore, Md.)  2013;58(1):108-119.
The intestinal mucus layer protects the epithelium from noxious agents, viruses, and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease requires translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. We investigated the roles of the intestinal mucus layer, and in particular Muc2, in development of experimental alcohol-associated liver disease in mice. We studied experimental alcohol-induced liver disease, induced by the Tsukamoto-French method (which involves continuous intragastric feeding of an isocaloric diet or alcohol) in wild-type and Muc2−/− mice. Muc2−/− mice showed less alcohol-induced liver injury and steatosis that developed in wild-type mice. Most notably, Muc2−/− mice had significantly lower plasma levels of lipopolysaccharide than wild-type mice after alcohol feeding. In contrast to wild-type mice, Muc2−/− mice were protected from alcohol-associated microbiome changes that are dependent on intestinal mucins. The anti-microbial proteins Reg3b and Reg3g were expressed at significantly higher levels in the jejunum of Muc2−/− mice fed the isocaloric diet or alcohol, compared with wild-type mice. Consequently, Muc2−/− mice showed increased killing of commensal bacteria and prevented intestinal bacterial overgrowth. Conclusion: Muc2−/− mice are protected from intestinal bacterial overgrowth and dysbiosis in response to alcohol feeding. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation, decreasing liver disease.
PMCID: PMC3695050  PMID: 23408358
microbiome; intestinal bacterial overgrowth; bacterial translocation; endotoxin; Reg3
4.  Atomic Layer Deposition to Fine-Tune the Surface Properties and Diameters of Fabricated Nanopores 
Nano letters  2004;4(7):1333-1337.
Atomic layer deposition of alumina enhanced the molecule sensing characteristics of fabricated nanopores by fine-tuning their surface properties, reducing 1/f noise, neutralizing surface charge to favor capture of DNA and other negative polyelectrolytes, and controlling the diameter and aspect ratio of the pores with near single Ångstrom precision. The control over the chemical and physical nature of the pore surface provided by atomic layer deposition produced a higher yield of functional nanopore detectors.
PMCID: PMC4076156  PMID: 24991194
5.  A Boswellic Acid-Containing Extract Ameliorates Schistosomiasis Liver Granuloma and Fibrosis through Regulating NF-κB Signaling in Mice 
PLoS ONE  2014;9(6):e100129.
Boswellic acid (BA)-containing extracts such as BSE have anti-inflammatory and immunomodulatory activity. In chronic schistosomiasis, the hepatic granuloma and fibrosis induced by egg deposition in the liver is the most serious pathological manifestations. However, little is known regarding the role of BAs in Schistosoma japonicum (S. japonicum) egg-induced liver granuloma and fibrosis. In order to investigate the effect of a water-soluble complex preparation of BSE, BSE-CD, on S. japonicum egg-induced liver pathology, liver granuloma and fibrosis were induced by infecting C57BL/6 mice with 18–22 cercariae of S. japonicum. S. japonicum cercariae infected mice were injected with BSE-CD at the onset of egg granuloma formation (early phase BSE-CD treatment after 4 weeks infection) or after the formation of liver fibrosis (late phase BSE-CD treatment after 7 weeks infection). Our data show that treatment of infected mice with BSE-CD significantly reduced both the extent of hepatic granuloma and fibrosis. Consistent with an inhibition of NF-κB signaling as evidenced by reduced IκB kinase (IKK) activation, the mRNA expression of VEGF (vascular endothelial growth factor, VEGF), TNF-α (tumor necrosis factor-alpha TNF-α) and MCP-1 (monocyte chemotactic protein 1, MCP-1) was decreased. Moreover, immunohistochemical analysis (IHC) revealed that the content of α-SMA in liver tissue of BSE-CD treated mice was dramatically decreased. Our findings suggest that BSE-CD treatment attenuates S. japonicum egg-induced hepatic granulomas and fibrosis, at least partly due to reduced NF-κB signaling and the subsequently decreased expression of VEGF, TNF-α, and MCP-1. Suppression of the activation of hepatic stellate cells (HSC) may also be involved in the therapeutic efficacy of BSE-CD.
PMCID: PMC4062494  PMID: 24941000
6.  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
7.  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
8.  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
9.  Comparative Ecophysiological Study of Salt Stress for Wild and Cultivated Soybean Species from the Yellow River Delta, China 
The Scientific World Journal  2014;2014:651745.
Osmotic and ionic stresses were the primary and instant damage produced by salt stress. They can also bring about other secondary stresses. Soybean is an important economic crop and the wild soybean aroused increasing attention for its excellent performance in salt resistance. For this reason, we compared the different performances of Glycine max L. (ZH13) and Glycine soja L. (BB52) in both young and mature seedlings, hoping to clarify the specific reasons. Our research revealed that, compared to the cultivated soybean, the wild soybean was able to maintain higher water potential and relative water content (RWC), accumulate more amount of proline and glycine betaine, reduce the contents of Na+ and Cl− by faster efflux, and cut down the efflux of the K+ as well as keep higher K+/Na+ ratio. And what is more is that, almost all the excel behaviors became particularly obvious under higher NaCl concentration (300 mM). Therefore, according to all the detections and comparisons, we concluded that the wild soybean had different tolerance mechanisms and better salt resistance. It should be used as eminent germplasm resource to enhance the resistant ability of cultivated soybean or even other crops.
PMCID: PMC4066866  PMID: 24999494
10.  Estrogen-Related Receptor Alpha Confers Methotrexate Resistance via Attenuation of Reactive Oxygen Species Production and P53 Mediated Apoptosis in Osteosarcoma Cells 
BioMed Research International  2014;2014:616025.
Osteosarcoma (OS) is a malignant tumor mainly occurring in children and adolescents. Methotrexate (MTX), a chemotherapy agent, is widely used in treating OS. However, treatment failures are common due to acquired chemoresistance, for which the underlying molecular mechanisms are still unclear. In this study, we report that overexpression of estrogen-related receptor alpha (ERRα), an orphan nuclear receptor, promoted cell survival and blocked MTX-induced cell death in U2OS cells. We showed that MTX induced ROS production in MTX-sensitive U2OS cells while ERRα effectively blocked the ROS production and ROS associated cell apoptosis. Our further studies demonstrated that ERRα suppressed ROS induction of tumor suppressor P53 and its target genes NOXA and XAF1 which are mediators of P53-dependent apoptosis. In conclusion, this study demonstrated that ERRα plays an important role in the development of MTX resistance through blocking MTX-induced ROS production and attenuating the activation of p53 mediated apoptosis signaling pathway, and points to ERRα as a novel target for improving osteosarcoma therapy.
PMCID: PMC4055217  PMID: 24967384
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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