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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.  Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis 
Cell Research  2014;24(10):1214-1230.
Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be achieved. Here we developed and applied a dual-fluorescence-based Protein Turnover Assay (ProTA) to quantitatively profile global changes in human protein degradome upon BTZ-induced proteasomal inhibition. ProTA and subsequent network analyses delineate potential molecular basis for BTZ action and tumor drug resistance in BTZ chemotherapy. Finally, combined use of BTZ with drugs targeting the ProTA-identified key genes or pathways in BTZ action reduced BTZ resistance in multiple myeloma cells. Remarkably, BTZ stabilizes proteasome subunit PSMC1 and proteasome assembly factor PSMD10, suggesting a previously under-appreciated mechanism for regulating proteasome homeostasis. Therefore, ProTA is a novel tool for profiling human protein degradome to elucidate potential mechanisms of drug action and resistance, which might facilitate therapeutic development targeting proteostasis to treat human disorders.
PMCID: PMC4185348  PMID: 25223703
bortezomib; combination therapy; proteostasis; protein degradome; proteasome homeostasis; N-end rule; ubiquitin
3.  Validation study of the modified injection technique for internal mammary sentinel lymph node biopsy in breast cancer 
OncoTargets and therapy  2015;8:2705-2708.
According to the hypothesis of internal mammary sentinel lymph node (IM-SLN) lymphatic drainage pattern, a modified radiotracer injection technique (periareolar intraparenchyma, high volume, and ultrasonographic guidance) was established. To verify the accuracy of the hypothesis and validate the modified radiotracer injection technique and to observe whether the lymphatic drainage of the whole breast parenchyma could reach to the same IM-SLN, different tracers were injected into different locations of the breast. The validation study results showed that the correlation and the agreement of the radiotracer and the fluorescence tracer are significant (case-base, rs =0.808, P<0.001; Kappa =0.79, P<0.001). It proved that the lymphatic drainage from different location of the breast (the primary tumor, the subareolar plexus) reached the same IM-SLNs and the hypothesis of IM-SLN lymphatic drainage pattern (ie, IM-SLN receives lymphatic drainage from not only the primary tumor area, but also the entire breast parenchyma). In other words, it validated the accuracy of our modified radiotracer injection technique.
PMCID: PMC4590635  PMID: 26445553
breast cancer; internal mammary; sentinel lymph node biopsy; visualization rate
4.  Increased Expression of Tissue/Salivary Transgelin mRNA Predicts Poor Prognosis in Patients with Oral Squamous Cell Carcinoma (OSCC) Surgery 
Transgelin is supposed to be a tumor suppression gene and it is down-regulated in a variety of human cancers. However, the role of transgelin in different cancers is still very controversial. In addition, currently little information is available the relationship between transgelin and Oral Squamous Cell Carcinoma (OSCC).
Western Blotting was performed to test the transgelin protein expression level in OSCC tissues and adjacent normal tissues. Real-time PCR was used to examine the expression level of transgelin mRNA in tissue, serum and saliva of OSCC patients and negative controls. The correlation between tissue and salivary transgelin mRNA expression level with a variety of clinical parameters was further studied.
Transgelin protein expression was increased in OSCC patients compared with healthy individuals. Similarly, the expression level of both tissue and salivary transgelin mRNA were increased significantly in patients with OSCC in comparison with normal controls. However, little difference of serum transgelin mRNA expression was found between the OSCC patients and healthy controls. In addition, overexpression of tissue or salivary transgelin was closely associated with various clinical parameters including poorer overall survival. Furthermore, our results showed that tissue and salivary transgelin mRNA were independent prognosis factors for OSCC.
The expressions level of tissue mRNA and protein were increased in OSCC patients. Both tissue and salivary transgelin mRNA were closely correlated with various important clinicopathological parameters and were independent prognosis factors for OSCC, indicating they might serve promising biomarkers for OSCC.
PMCID: PMC4530988  PMID: 26242444
Mouth Neoplasms; RNA, Messenger; Saliva; Serum
5.  Sympathetic Nerve Fibers in Human Cervical and Thoracic Vagus Nerves 
Vagus nerve stimulation therapy (VNS) has been used for chronic heart failure (CHF), and is believed to improve imbalance of autonomic control by increasing parasympathetic activity. Although it is known that there is neural communication between the VN and the cervical sympathetic trunk, there are few data regarding the quantity and/or distribution of the sympathetic components within the VN.
To examine the sympathetic component within human VN and correlate these with the presence of cardiac and neurologic diseases.
We performed immunohistochemistry on 31 human cervical and thoracic VNs (total 104 VNs) from autopsies and we reviewed the patients’ records. We correlated the quantity of sympathetic nerve fibers within the VNs with cardiovascular and neurologic disease states.
All 104 VNs contain TH positive (sympathetic) nerve fibers; the mean TH positive areas were 5.47% in right cervical, 3.97% in left cervical, 5.11% in right thoracic, and 4.20% in left thoracic VN. The distribution of TH positive nerve fibers varied from case to case: central, peripheral, or scattered throughout nerve bundles. No statistically significant differences in nerve morphology were seen between diseases in which VNS is considered effective (depression and CHF), and other cardiovascular diseases, or neurodegenerative disease.
Human VNs contain sympathetic nerve fibers. The sympathetic component within the VN could play a role in physiologic effects reported with VNS. The recognition of sympathetic nerve fibers in the VNs may lead to better understanding of the therapeutic mechanisms of VNS.
PMCID: PMC4108556  PMID: 24768897
Cervical vagus nerves; Sympathetic nerves; Ganglion cells; Heart failure; Vagal nerve stimulation
6.  Selective Sinoatrial Node Optical Mapping and the Mechanism of Sinus Rate Acceleration 
Studies using isolated sinoatrial node (SAN) cells indicate that rhythmic spontaneous sarcoplasmic reticulum calcium release (Ca clock) plays an important role in SAN automaticity. In the intact SAN, cross contamination of optical signals from the SAN and the right atrium (RA) prevented the definitive testing of Ca clock hypothesis. The aim of this study was to use a novel approach to selectively map intact SAN to examine the Ca clock mechanism.
Methods and Results
We simultaneously mapped intracellular Ca (Cai) and membrane potential (Vm) in 10 isolated, Langendorff-perfused normal canine RAs. The excitability of the RA was suppressed with high potassium Tyrode’s solution, allowing selective optical mapping of Vm and Cai of the SAN. Isoproterenol (ISO, 0.03 µmol/L) decreased cycle length of the sinus beats, and shifted the leading pacemaker site from the middle or inferior SAN to the superior SAN in all RAs. The Cai upstroke preceded the Vm in the leading pacemaker site by up to 18±2 ms. ISO-induced changes to SAN were inhibited by ryanodine (3 µmol/L), but not ZD7288 (3 µmol/L), a selective If blocker.
We conclude that, in isolated canine right atrium, high extracellular potassium concentration can suppress atrial excitability thus leading to SAN-RA conduction block, allowing selective optical mapping of the intact SAN. Acceleration of calcium cycling in the superior SAN underlies the mechanism of sinus tachycardia during sympathetic stimulation.
PMCID: PMC4506937  PMID: 22094913
calcium; nervous system; sympathetic; potassium; sarcoplasmic reticulum; sinoatrial node
7.  Concentration- and chromosome-organization-dependent regulator unbinding from DNA for transcription regulation in living cells 
Nature Communications  2015;6:7445.
Binding and unbinding of transcription regulators at operator sites constitute a primary mechanism for gene regulation. While many cellular factors are known to regulate their binding, little is known on how cells can modulate their unbinding for regulation. Using nanometer-precision single-molecule tracking, we study the unbinding kinetics from DNA of two metal-sensing transcription regulators in living Escherichia coli cells. We find that they show unusual concentration-dependent unbinding kinetics from chromosomal recognition sites in both their apo and holo forms. Unexpectedly, their unbinding kinetics further varies with the extent of chromosome condensation, and more surprisingly, varies in opposite ways for their apo-repressor versus holo-activator forms. These findings suggest likely broadly relevant mechanisms for facile switching between transcription activation and deactivation in vivo and in coordinating transcription regulation of resistance genes with the cell cycle.
Binding and unbinding of transcription regulators at operator sites regulates gene expression. By single-molecule tracking of metal-sensing regulators, here the authors show that the unbinding kinetics depends on regulator concentration and chromosome condensation, and varies with their metal-binding states.
PMCID: PMC4507017  PMID: 26145755
8.  Ubiquitylation of Autophagy Receptor Optineurin by HACE1 Activates Selective Autophagy for Tumor Suppression 
Cancer cell  2014;26(1):106-120.
In selective autophagy, receptors are central for cargo selection and delivery. However, it remains yet unclear whether and how multiple autophagy receptors might form complex and function concertedly to control autophagy. Optineurin (OPTN), implicated genetically in glaucoma and amyotrophic lateral sclerosis, was a recently identified autophagy receptor. Here we report that tumor suppressor HACE1, a ubiquitin ligase, ubiquitylates OPTN and promotes its interaction with p62/SQSTM1 to form the autophagy receptor complex, thus accelerating autophagic flux. Interestingly, the K48-linked polyubiquitin chains that HACE1 conjugates onto OPTN might predominantly target OPTN for autophagic degradation. By demonstrating that the HACE1-OPTN axis synergistically suppresses growth and tumorigenicity of lung cancer cells, our findings may open an avenue for developing autophagy-targeted therapeutic intervention into cancer.
PMCID: PMC4166492  PMID: 25026213
9.  Concise Synthesis of Functionalized Benzocyclobutenones 
Tetrahedron  2014;70(27-28):4135-4146.
A concise approach to access functionalized benzocyclobutenones from 3-halophenol derivatives is described. This modified synthesis employs a [2+2] cycloaddition between benzynes generated from dehydrohalogenation of aryl halides using LiTMP and acetaldehyde enolate generated from n-BuLi and THF, followed by oxidation of the benzocyclobutenol intermediates to provide benzocyclobutenones. The [2+2] reaction can be run on a 10-gram scale with an increased yield. A number of functional groups including alkenes and alkynes are tolerated. Coupling of benzynes with ketene silyl acetals to give 8-substituted benzocyclobutenones is also demonstrated.
PMCID: PMC4049219  PMID: 24926108
benzocyclobutenone; cycloaddition; benzyne; dehydrohalogenation; benzocyclobutenol
10.  Aging and Exercise Affect Hippocampal Neurogenesis via Different Mechanisms 
PLoS ONE  2015;10(7):e0132152.
The rate of neurogenesis is determined by 1) the number of neural stem/progenitor cells (NSCs), 2) proliferation of NSCs, 3) neuron lineage specification, and 4) survival rate of the newborn neurons. Aging lowers the rate of hippocampal neurogenesis, while exercise (Ex) increases this rate. However, it remains unclear which of the determinants are affected by aging and Ex. We characterized the four determinants in different age groups (3, 6, 9, 12, 21 months) of mice that either received one month of Ex training or remained sedentary. Bromodeoxyuridine (BrdU) was injected two hours before sacrificing the mice to label the proliferating cells. The results showed that the number of newborn neurons massively decreased (>95%) by the time the mice reached nine months of age. The number of NSC was mildly reduced during aging, while Ex delayed such decline. The proliferation rates were greatly decreased by the time the mice were 9-month-old and Ex could not improve the rates. The rates of neuron specification were decreased during aging, while Ex increased the rates. The survival rate was not affected by age or Ex. Aging greatly reduced newborn neuron maturation, while Ex potently enhanced it. In conclusion, age-associated decline of hippocampal neurogenesis is mainly caused by reduction of NSC proliferation. Although Ex increases the NSC number and neuron specification rates, it doesn't restore the massive decline of NSC proliferation rate. Hence, the effect of Ex on the rate of hippocampal neurogenesis during aging is limited, but Ex does enhance the maturation of newborn neurons.
PMCID: PMC4493040  PMID: 26147302
11.  Effects of carvedilol on cardiac autonomic nerve activities during sinus rhythm and atrial fibrillation in ambulatory dogs 
Europace  2014;16(7):1083-1091.
We hypothesized that carvedilol can effectively suppress autonomic nerve activity (ANA) in ambulatory dogs during sinus rhythm and atrial fibrillation (AF), and that carvedilol withdrawal can lead to rebound elevation of ANA. Carvedilol is known to block pre-junctional β2-adrenoceptor responsible for norepinephrine release.
Methods and results
We implanted radiotransmitters to record stellate ganglion nerve activity (SGNA), vagal nerve activity (VNA), and superior left ganglionated plexi nerve activity (SLGPNA) in 12 ambulatory dogs. Carvedilol (12.5 mg orally twice a day) was given for 7 days during sinus rhythm (n = 8). Four of the eight dogs and an additional four dogs were paced into persistent AF. Carvedilol reduced heart rate [from 103 b.p.m. (95% confidence interval (CI), 100–105) to 100 b.p.m. (95% CI, 98–102), P = 0.044], suppressed integrated nerve activities (Int-NAs, SGNA by 17%, VNA by 19%, and SLGPNA by 12%; all P < 0.05 vs. the baseline), and significantly reduced the incidence (from 8 ± 6 to 3 ± 3 episodes/day, P < 0.05) and total duration (from 68 ± 64 to 16 ± 21 s/day, P < 0.05) of paroxysmal atrial tachycardia (PAT). Following the development of persistent AF, carvedilol loading was associated with AF termination in three dogs. In the remaining five dogs, Int-NAs were not significantly suppressed by carvedilol, but SGNA significantly increased by 16% after carvedilol withdrawal (P < 0.001).
Carvedilol suppresses ANA and PAT in ambulatory dogs during sinus rhythm.
PMCID: PMC4081622  PMID: 24469435
Autonomic nervous system; Atrium; Arrhythmia; Atrial fibrillation; Carvedilol
12.  Multiple Nonglycemic Genomic Loci Are Newly Associated With Blood Level of Glycated Hemoglobin in East Asians 
Diabetes  2014;63(7):2551-2562.
Glycated hemoglobin A1c (HbA1c) is used as a measure of glycemic control and also as a diagnostic criterion for diabetes. To discover novel loci harboring common variants associated with HbA1c in East Asians, we conducted a meta-analysis of 13 genome-wide association studies (GWAS; N = 21,026). We replicated our findings in three additional studies comprising 11,576 individuals of East Asian ancestry. Ten variants showed associations that reached genome-wide significance in the discovery data set, of which nine (four novel variants at TMEM79 [P value = 1.3 × 10−23], HBS1L/MYB [8.5 × 10−15], MYO9B [9.0 × 10−12], and CYBA [1.1 × 10−8] as well as five variants at loci that had been previously identified [CDKAL1, G6PC2/ABCB11, GCK, ANK1, and FN3KI]) showed consistent evidence of association in replication data sets. These variants explained 1.76% of the variance in HbA1c. Several of these variants (TMEM79, HBS1L/MYB, CYBA, MYO9B, ANK1, and FN3K) showed no association with either blood glucose or type 2 diabetes. Among individuals with nondiabetic levels of fasting glucose (<7.0 mmol/L) but elevated HbA1c (≥6.5%), 36.1% had HbA1c <6.5% after adjustment for these six variants. Our East Asian GWAS meta-analysis has identified novel variants associated with HbA1c as well as demonstrated that the effects of known variants are largely transferable across ethnic groups. Variants affecting erythrocyte parameters rather than glucose metabolism may be relevant to the use of HbA1c for diagnosing diabetes in these populations.
PMCID: PMC4284402  PMID: 24647736
13.  Transporting Cells in Semi-Solid Gel Condition and at Ambient Temperature 
PLoS ONE  2015;10(6):e0128229.
Mammalian cells including human cancer cells are usually transported in cryovials on dry ice or in a liquid nitrogen vapor shipping vessel between different places at long distance. The hazardous nature of dry ice and liquid nitrogen, and the associated high shipping cost strongly limit their routine use. In this study, we tested the viability and properties of cells after being preserved or shipped over long distance in Matrigel mixture for different days. Our results showed that cells mixed with Matrigel at suitable ratios maintained excellent viability (>90%) for one week at room temperature and preserved the properties such as morphology, drug sensitivity and metabolism well, which was comparable to cells cryopreserved in liquid nitrogen. We also sent cells in the Matrigel mixture via FedEx service to different places at ambient temperature. Upon arrival, it was found that over 90% of the cells were viable and grew well after replating. These data collectively suggested that our Matrigel-based method was highly convenient for shipping live cells for long distances in semi-solid gel condition and at ambient temperature.
PMCID: PMC4476595  PMID: 26098554
14.  Polymorphisms and plasma levels of IL-27: impact on genetic susceptibility and clinical outcome of bladder cancer 
BMC Cancer  2015;15:433.
Interleukin-27 (IL-27) has been recognized as a pleiotropic cytokine with both pro- and anti-inflammatory properties. Few studies have investigated polymorphisms and serum/plasma levels of IL-27 in diseases including cancers. This study has analyzed the associations of IL-27 gene polymorphisms, as well as plasma levels of IL-27, with susceptibility to bladder cancer and clinical outcome.
Three hundred and thirty-two patients (nonmuscle-invasive bladder cancer (NMIBC)/muscle-invasive bladder cancer (MIBC): 176/156) included in a 60-month follow-up program and 499 controls were enrolled. Two single nucleotide polymorphisms (SNPs), rs153109 and rs17855750, were genotyped by polymerase chain reaction (PCR) -restriction fragment length polymorphism (RFLP) method. Plasma concentration of IL-27 was determined by ELISA in 124 patients (NMIBC/MIBC: 50/74) and 151 controls.
Significantly increased risk for bladder cancer was associated with AG/GG genotypes of rs153109 (P = 0.029). No GG genotype of rs17855750 was observed in controls, while 4 patients were found to be GG homozygotes, suggesting GG genotype may be associated with bladder cancer risk (P = 0.006). For bladder cancer patients, SNP rs17855750 was also associated with increased risk for MIBC. For MIBC patients, but not NMIBC, TG/GG genotypes of rs17855750 turned out to be a protective factor for overall survival (P = 0.035). Significantly reduced plasma levels of IL-27 were observed in both NMIBC and MIBC patients compared with controls (P < 0.0001).
Our data suggest that polymorphisms and reduced plasma levels of IL-27 may predict the susceptibility to bladder cancer, and rs17855750 may be a useful marker to distinguish patients with high risk of death.
PMCID: PMC4445811  PMID: 26014498
Bladder cancer; IL-27; Polymorphisms; Plasma levels; Susceptibility; Prognosis
15.  Benign metastasizing leiomyoma of the lung: A case report and literature review 
Oncology Letters  2015;10(1):307-312.
Pulmonary benign metastasizing leiomyoma (BML) is a rare event characterized by benign soft-tissue tumors that occur when uterine leiomyomas metastasize to the lung. The present study reports the case of a 47-year-old female patient who presented with multiple bilateral pulmonary nodules on a chest X-ray during a health checkup nine years after a hysterectomy due to uterine fibroids. Chest computed tomography (CT) revealed multiple well-defined nodular shadows in the lung. One tumor of the left upper lung was resected by thoracoscopic surgery. Pathologically, the resected lesion consisted of benign spindle cells and was diagnosed as BML. The post-operative course was uneventful. Other lung nodules have been meticulously monitored at follow-up, and repeat CT two years later showed that these nodules had not increased at all in size and that no new lobe nodules had appeared. The present study indicates that pulmonary BML occurs in a low proportion of female with a history of uterine leiomyoma and treatment methods for it are diverse and controversial.
PMCID: PMC4487159  PMID: 26171020
benign metastasizing leiomyoma; pulmonary nodules
16.  Advanced glycation end-product expression is upregulated in the gastrointestinal tract of type 2 diabetic rats 
World Journal of Diabetes  2015;6(4):662-672.
AIM: To investigate changes in advanced glycation end products (AGEs) and their receptor (RAGE) expression in the gastrointestinal (GI) tract in type 2 diabetic rats.
METHODS: Eight inherited type 2 diabetic rats Goto-Kakizak (GK) and ten age-matched normal rats were used in the study. From 18 wk of age, the body weight and blood glucose were measured every week and 2 wk respectively. When the rats reached 32 wk, two-centimeter segments of esophagus, duodenum, jejunum, ileum, and colon were excised and the wet weight was measured. The segments were fixed in 10% formalin, embedded in paraffin and five micron sections were cut. The layer thickness was measured in Hematoxylin and Eosin-stained slides. AGE [N epsilon-(carboxymethyl) lysine and N epsilon-(carboxyethyl)lysine] and RAGE were detected by immunohistochemistry staining and image analysis was done using Sigmascan Pro 4.0 image analysis software.
RESULTS: The blood glucose concentration (mmol/L) at 18 wk age was highest in the GK group (8.88 ± 1.87 vs 6.90 ± 0.43, P < 0.001), a difference that continued to exist until the end of the experiment. The wet weight per unit length (mg/cm) increased in esophagus, jejunum and colon from the normal to the GK group (60.64 ± 9.96 vs 68.56 ± 11.69, P < 0.05 for esophagus; 87.01 ± 9.35 vs 105.29 ± 15.45, P < 0.01 for jejunum; 91.37 ± 7.25 vs 97.28 ± 10.90, P < 0.05 for colon). Histologically, the layer thickness of the GI tract was higher for esophagus, jejunum and colon in the GK group [full thickness (μm): 575.37 ± 69.22 vs 753.20 ± 150.41, P < 0.01 for esophagus; 813.51 ± 44.44 vs 884.81 ± 45.31, P < 0.05 for jejunum; 467.12 ± 65.92 vs 572.26 ± 93.60, P < 0.05 for colon]. In esophagus, the AGE and RAGE mainly distributed in striated muscle cells and squamous epithelial cells. The AGE distribution was much stronger in the GK group compared to the normal group both in the striated muscle layer and mucosa layer (immuno-positive area/ total measuring area %: 4.52 ± 0.89 vs 10.96 ± 1.34, P < 0.01 for muscle; 8.90 ± 2.62 vs 22.45 ± 1.26, P < 0.01 for mucosa). No visible difference was found for RAGE distribution between the two groups. In the intestine AGE and RAGE distributed in epithelial cells of villi and crypt. RAGE was also found in neurons in the myenteric and submucosal plexus. The intensity of AGE staining in mucosa of all segments and RAGE staining in neurons in all segments were strongest in the diabetes group. Significant difference for AGE was found in the epithelial cells of villi and crypt in duodenum (immuno-positive area/total measuring area %: 13.37 ± 3.51 vs 37.48 ± 8.43, P < 0.05 for villi; 0.38 ± 0.12 vs 1.87 ± 0.53, P < 0.05 for crypt) and for RAGE in neurons of all segments (e.g., for jejunum: no staining neurons% 0 vs 0, mild 36.0 ± 5.2 vs 28.7 ± 3.5, moderate 53.2 ± 4.8 vs 55.8 ± 5.4, strong 10.7 ± 1.1 vs 15.4 ± 2.0, P < 0.05). In the colon, RAGE was primarily found in neurons in the myenteric and submucosal plexus. It was stronger in the diabetes group than in the normal group (no staining neurons% 6.2 ± 0.2 vs 0.3 ± 0.04, mild 14.9 ± 2.1 vs 17.6 ± 1.5, moderate 53.1 ± 4.6 vs 44.7 ± 4.4, strong 25.6 ± 18 vs 43.6 ± 4.0, P < 0.05). In the rectum, RAGE was primarily found in the mucosa epithelial cells.
CONCLUSION: The AGE and RAGE expression was up-regulated in the GI tract of GK diabetic rats and may contribute to GI dysfunction in type 2 diabetic patients.
PMCID: PMC4434088  PMID: 25987965
Diabetes mellitus; Gastrointestinal complications; Advanced glycation end products; Receptor of advanced glycation end products
17.  Systemic lupus erythematosus accompanying with renal tuberculosis: a case report 
A 26-year-old woman, with a six-year history of well-controlled systemic lupus erythematosus (SLE), complained of urinary frequency and urgency. After failure of commonly-used antibiotic therapy, mycobacterium tuberculosis was cultured from her urine and renal tuberculosis (TB) was diagnosed. However, she underwent right nephrectomy after the combination therapies of prednisone for SLE and anti-tuberculosis treatment for renal TB failed. To our knowledge, SLE accompanying renal TB is rare, and such a rapid deterioration in renal function has never been reported.
PMCID: PMC4509340  PMID: 26221395
Systemic lupus erythematosus; renal tuberculosis; non-functioning kidney
18.  Expiratory flow limitation relates to symptoms during COPD exacerbations requiring hospital admission 
Expiratory flow limitation (EFL) is seen in some patients presenting with a COPD exacerbation; however, it is unclear how EFL relates to the clinical features of the exacerbation. We hypothesized that EFL when present contributes to symptoms and duration of recovery during a COPD exacerbation. Our aim was to compare changes in EFL with symptoms in subjects with and without flow-limited breathing admitted for a COPD exacerbation.
Subjects and methods
A total of 29 subjects with COPD were recruited within 48 hours of admission to West China Hospital for an acute exacerbation. Daily measurements of post-bronchodilator spirometry, resistance, and reactance using the forced oscillation technique and symptom (Borg) scores until discharge were made. Flow-limited breathing was defined as the difference between inspiratory and expiratory respiratory system reactance (EFL index) greater than 2.8 cmH2O·s·L−1. The physiological predictors of symptoms during recovery were determined by mixed-effect analysis.
Nine subjects (31%) had flow-limited breathing on admission despite similar spirometry compared to subjects without flow-limited breathing. Spirometry and resistance measures did not change between enrolment and discharge. EFL index values improved in subjects with flow-limited breathing on admission, with resolution in four patients. In subjects with flow-limited breathing on admission, symptoms were related to inspiratory resistance and EFL index values. In subjects without flow-limited breathing, symptoms related to forced expiratory volume in 1 second/forced vital capacity. In the whole cohort, EFL index values at admission was related to duration of stay (Rs=0.4, P=0.03).
The presence of flow-limited breathing as well as abnormal respiratory system mechanics contribute independently to symptoms during COPD exacerbations.
PMCID: PMC4437522  PMID: 25999709
COPD; exacerbations; flow limitation; respiratory mechanics; symptoms
20.  Feasibility of biohydrogen production from industrial wastes using defined microbial co-culture 
Biological Research  2015;48(1):24.
The development of clean or novel alternative energy has become a global trend that will shape the future of energy. In the present study, 3 microbial strains with different oxygen requirements, including Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, were used to construct a hydrogen production system that was composed of a mixed aerobic-facultative anaerobic-anaerobic consortium. The effects of metal ions, organic acids and carbohydrate substrates on this system were analyzed and compared using electrochemical and kinetic assays. It was then tested using small-scale experiments to evaluate its ability to convert starch in 5 L of organic wastewater into hydrogen. For the one-step biohydrogen production experiment, H1 medium (nutrient broth and potato dextrose broth) was mixed directly with GAM broth to generate H2 medium (H1 medium and GAM broth). Finally, Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D of three species microbial co-culture to produce hydrogen under anaerobic conditions. For the two-step biohydrogen production experiment, the H1 medium, after cultured the microbial strains Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, was centrifuged to remove the microbial cells and then mixed with GAM broth (H2 medium). Afterward, the bacterial strain Clostridium acetobutylicum ATCC 824 was inoculated into the H2 medium to produce hydrogen by anaerobic fermentation.
The experimental results demonstrated that the optimum conditions for the small-scale fermentative hydrogen production system were at pH 7.0, 35°C, a mixed medium, including H1 medium and H2 medium with 0.50 mol/L ferrous chloride, 0.50 mol/L magnesium sulfate, 0.50 mol/L potassium chloride, 1% w/v citric acid, 5% w/v fructose and 5% w/v glucose. The overall hydrogen production efficiency in the shake flask fermentation group was 33.7 mL/h-1.L-1, and those the two-step and the one-step processes of the small-scale fermentative hydrogen production system were 41.2 mL/h-1.L-1 and 35.1 mL/h-1.L-1, respectively.
Therefore, the results indicate that the hydrogen production efficiency of the two-step process is higher than that of the one-step process.
PMCID: PMC4427975  PMID: 25943991
Renewable Energy; Biohydrogen; Microbial consortium; Hydrogen
21.  Carvedilol Analogue Modulates both Basal and Stimulated Sinoatrial Node Automaticity 
Heart and vessels  2013;29(3):396-403.
The membrane voltage clock and calcium (Ca2+) clock jointly regulate sinoatrial node (SAN) automaticity. VK-II-36 is a novel carvedilol analog that suppress sarcoplasmic reticulum (SR) Ca2+ release but does not block β-receptor. The effect of VK-II-36 on SAN function remains unclear. The purpose of this study was to evaluate whether VK-II-36 can influence SAN automaticity through inhibiting the Ca2+ clock.
Methods and Results
We simultaneously mapped intracellular Ca2+ and membrane potential in 24 isolated canine right atriums, using previously described criteria of the timing of late diastolic intracellular Ca elevation (LDCAE) relative to the action potential upstroke to detect the Ca2+ clock. Pharmacological intervention with isoproterenol (ISO), ryanodine, caffeine, and VK-II-36 were performed after baseline recordings. VK-II-36 caused sinus rate downregulation and reduced LDCAE in the pacemaking site under basal condition (P<0.01). ISO induced an upward shift of the pacemaking site in SAN and augmented LDCAE in pacemaking site. ISO also significantly and dose-dependently increased the sinus rate. The treatment of VK-II-36 (30 μmol/L) abolished both the ISO-induced shift of pacemaking site and augmentation of LDCAE (P<0.01), and suppressed the ISO-induced increase in sinus rate (P=0.02).
Our results suggest that sinus rate may be partly controlled by Ca2+ clock via SR Ca2+ release during β-adrenergic stimulation.
PMCID: PMC3849120  PMID: 23836067
calcium; sympathetic nervous system; sinoatrial node; sarcoplasmic reticulum; store-overload-induced Ca release
22.  Utilization Rates of Implantable Cardioverter-Defibrillators for Primary Prevention of Sudden Cardiac Death: A 2012 Calculation for a Midwestern Health Referral Region 
Utilization rates (URs) for implantable cardioverter-defibrillators (ICDs) for primary prevention of sudden cardiac death (PPSCD) are lacking in the community.
To establish the ICD UR in central Indiana.
A query run on two hospitals in a health information exchange database in Indianapolis identified patients between 2011 and 2012 with left ventricular ejection fraction (EF) ≤0.35. ICD-eligibility and utilization were determined from chart review.
We identified 1,863 patients with at least one low-EF study. Two cohorts were analyzed: 1,672 patients without, and 191 patients with, ICD-9-CM procedure code 37.94 for ICD placement. We manually reviewed a stratified (by hospital) random sample of 300 patients from the no-ICD procedure code cohort and found that 48 (16%) had no ICD but had class I indications for ICD. Eight of 300 (2.7%) actually had ICD implantation for PPSCD. Review of all 191 patients in the ICD procedure code cohort identified 70 with ICD implantation for PPSCD. The ICD UR (ratio between patients with ICD for PPSCD and all with indication) was 38% overall (95% CI 28–49%). URs were 48% for males (95% CI 34–61%), 21% for females (95% CI 16–26%, p=0.0002 vs males), 40% for whites (95% CI 27–53%), and 37% for blacks (95% CI 28–46%, p=0.66 vs whites).
The ICD UR is 38% among patients meeting Class I indications, suggesting further opportunities to improve guideline compliance. Furthermore, this study illustrates limitations in calculating ICD UR using large electronic repositories without hands-on chart review.
PMCID: PMC4074531  PMID: 24566233
defibrillator; implantable cardioverter-defibrillator; sudden cardiac death; arrhythmia; utilization
23.  Prohibitin Is Involved in Patients with IgG4 Related Disease 
PLoS ONE  2015;10(5):e0125331.
IgG4-related disease (IgG4-RD) is a chronic systemic disease involved in many organs and tissues. As only limited autoantigens have been found since the beginning of this century, the aim of this study was to reveal new candidate autoantigens of IgG4-RD.
Multiple cell lines including HT-29, EA.hy926, HEK 293 and HepG2 were used to test the binding ability of circulating autoantibodies from IgG4-RD sera. The amino-acid sequence was then analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF/TOF) mass spectrometry. After the cloning and expression of recombinant putative autoantigen in a bacterial expression system, the corresponding immuno assay was set up and utilized to observe the prevalence of serum autoantibodies in a large set of confirmed clinical samples.
One positive autoantigen was identified as prohibitin. ELISA analysis showed that a majority of patients with IgG4-RD have antibodies against prohibitin. Anti-prohibitin antibodies were present in the sera of patients with definite autoimmune pancreatitis (25/34; 73.5%), Mikulicz’s disease (8/15; 53.3%), retroperitoneal fibrosis (6/11; 54.5%), other probable IgG4-RD (26/29; 89.7%) and Sjögren’s syndrome (4/30; 13.3%) but not in apparently healthy donors (1/70; 1.4%).
An association between prohibitin and patients with some IgG4-RD was observed, although the results were quite heterogeneous among different individuals within autoimmune pancreatitis, Mikulicz’s disease and retroperitoneal fibrosis.
PMCID: PMC4416882  PMID: 25932630
24.  Function and Dysfunction of Human Sinoatrial Node 
Korean Circulation Journal  2015;45(3):184-191.
Sinoatrial node (SAN) automaticity is jointly regulated by a voltage (cyclic activation and deactivation of membrane ion channels) and Ca2+ clocks (rhythmic spontaneous sarcoplasmic reticulum Ca2+ release). Using optical mapping in Langendorff-perfused canine right atrium, we previously demonstrated that the β-adrenergic stimulation pushes the leading pacemaker to the superior SAN, which has the fastest activation rate and the most robust late diastolic intracellular calcium (Cai) elevation. Dysfunction of the superior SAN is commonly observed in animal models of heart failure and atrial fibrillation (AF), which are known to be associated with abnormal SAN automaticity. Using the 3D electroanatomic mapping techniques, we demonstrated that superior SAN served as the earliest atrial activation site (EAS) during sympathetic stimulation in healthy humans. In contrast, unresponsiveness of superior SAN to sympathetic stimulation was a characteristic finding in patients with AF and SAN dysfunction, and the 3D electroanatomic mapping technique had better diagnostic sensitivity than corrected SAN recovery time testing. However, both tests have significant limitations in detecting patients with symptomatic sick sinus syndrome. Recently, we reported that the location of the EAS can be predicted by the amplitudes of P-wave in the inferior leads. The inferior P-wave amplitudes can also be used to assess the superior SAN responsiveness to sympathetic stimulation. Inverted or isoelectric P-waves at baseline that fail to normalize during isoproterenol infusion suggest SAN dysfunction. P-wave morphology analyses may be helpful in determining the SAN function in patients at risk of symptomatic sick sinus syndrome.
PMCID: PMC4446811  PMID: 26023305
Calcium; Sinoatrial node; Adrenergic beta-agonists; Sick sinus syndrome; Biological pacemaker
25.  Electron Transfer Flavoprotein Subunit Beta Is a Candidate Endothelial Cell Autoantigen in Behçet’s Disease 
PLoS ONE  2015;10(4):e0124760.
Behçet’s disease (BD) is a chronic inflammatory disease with multisystem involvement, and it is listed as a rare disease in the United States but is common in the Middle East, China, and Japan. The aim of this study was to identify novel autoantigens in Chinese patients with BD. First, the candidate autoantigens were screened by Western blotting, and the sequences of putative antigens were identified by LC-MALDI-TOF/TOF mass spectrometry. Next, the screened protein was cloned, expressed and purified. Then, an optimized ELISA was developed, and the serological criteria were evaluated using a large number of confirmed patients. One antigen with a molecular weight of approximately 28 kDa was identified as electron transfer flavoprotein subunit beta (ETFB). Positive reactivity was detected in recombinant human ETFB sera from 38 of 92 BD patients (41 %) and 1 of 90 healthy controls (1 %).
PMCID: PMC4410958  PMID: 25915519

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