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1.  A Novel Mutation in the TECTA Gene in a Chinese Family with Autosomal Dominant Nonsyndromic Hearing Loss 
PLoS ONE  2014;9(2):e89240.
TECTA-related deafness can be inherited as autosomal-dominant nonsyndromic deafness (designated DFNA) or as the autosomal-recessive version. The α-tectorin protein, which is encoded by the TECTA gene, is one of the major components of the tectorial membrane in the inner ear. Using targeted DNA capture and massively parallel sequencing (MPS), we screened 42 genes known to be responsible for human deafness in a Chinese family (Family 3187) in which common deafness mutations had been ruled out as the cause, and identified a novel mutation, c.257–262CCTTTC>GCT (p. Ser86Cys; p. Pro88del) in exon 3 of the TECTA gene in the proband and his extended family. All affected individuals in this family had moderate down-sloping hearing loss across all frequencies. To our knowledge, this is the second TECTA mutation identified in Chinese population. This study demonstrates that targeted genomic capture, MPS, and barcode technology might broaden the availability of genetic testing for individuals with undiagnosed DFNA.
doi:10.1371/journal.pone.0089240
PMCID: PMC3931719  PMID: 24586623
2.  Rational Improvement of Simvastatin Synthase Solubility in Escherichia coli Leads to Higher Whole-cell Biocatalytic Activity 
Biotechnology and bioengineering  2009;102(1):20-28.
Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed ~50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 hours, which represents a significant improvement over the performance of wild type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes.
doi:10.1002/bit.22028
PMCID: PMC3928684  PMID: 18988191
Acyltransferase; simvastatin; site-directed mutagenesis; whole-cell biocatalyst; protein solubility
3.  Genetic mutations in nonsyndromic deafness patients of Chinese minority and han ethnicities in Yunnan, China 
Background
Each year in China, 30,000 babies are born with congenital hearing impairment. However, the molecular etiology of hearing impairment in the Yunnan Province population where more than 52 minorities live has not been thoroughly investigated. To provide appropriate genetic testing and counseling to these families, we investigated the molecular etiology of nonsyndromic deafness in this population.
Methods
Unrelated students with hearing loss (n = 235) who attended Kunming Huaxia secondary specialized school in Yunnan enrolled in this study. Three prominent deafness-related genes, GJB2, SLC26A4 and mtDNA 12S rRNA, were analyzed. High-resolution temporal bone computed tomography (CT) scan examinations were performed in 100 cases, including 16 cases with SLC26A4 gene variants, and 37 minorities and 47 Han cases without any SLC26A4 gene mutation.
Results
The GJB2 mutation was detected in 16.67% (7/42) of minority patients and 17.62% (34/193) of Chinese Han patients (P > 0.05). 235delC was the hotspot mutation in nonsyndromic hearing loss (NSHL) patients, whereas 35delG was not found. The 431_450del19 mutation was detected for the first time in Han NSHL patients, which resulted in a premature stop codon and changed the protein. The SLC26A4 mutation was found in 9.52% (4/42) of minority patients and 9.84% (19/193) of Han Chinese patients (P > 0.05). The frequencies of mtDNA 12S rRNA mutation in minority and Han Chinese patients were 11.90% (5/42) and 7.77% (15/193; P > 0.05), respectively. Sixteen (16/23, 69.57%) patients with SLC26A4 mutations received temporal bone CT scan, and 14 patients were diagnosed with enlarged vestibular aqueducts (EVAs); the other 2 patients had normal inner ear development. The ratio of EVA in the minorities was 14.63% (6/41).
Conclusions
In this study, a total of 35.74% deaf patients showed evidence of genetic involvement, based on either genetic screening or family history; 17.45%, 9.79%, and 8.51% of the patients were determined to have inherited hearing impairment caused by GJB2, SLC26A4, and mtDNA 1555A > G mutations. There was no significant difference in deafness associated gene mutational spectrum and frequency between the Yunnan minority and Han patients.
doi:10.1186/1479-5876-11-312
PMCID: PMC3878508  PMID: 24341454
Genetic mutations; Nonsyndromic deafness; Minority; Han; Yunnan
4.  High Glucose Induced Oxidative Stress and Apoptosis in Cardiac Microvascular Endothelial Cells Are Regulated by FoxO3a 
PLoS ONE  2013;8(11):e79739.
Aim
Cardiac microvascular endothelial cells (CMECs) dysfunction contributes to cardiovascular complications in diabetes, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors are involved in apoptosis and reactive oxygen species (ROS) production. Therefore, the present study was designed to elucidate the potential role of FoxO3a on the CMECs injury induced by high glucose.
Materials and Methods
CMECs were isolated from hearts of adult rats and cultured in normal or high glucose medium for 6 h, 12 h and 24 h respectively. To down-regulate FoxO3a expression, CMECs were transfected with FoxO3a siRNA. ROS accumulation and apoptosis in CMECs were assessed by dihydroethidine (DHE) staining and TUNEL assay respectively. Moreover, the expressions of Akt, FoxO3a, Bim and BclxL in CMECs were assessed by Western blotting assay.
Results
ROS accumulation in CMECs was significantly increased after high glucose incubation for 6 to 24 h. Meanwhile, high glucose also increased apoptosis in CMECs, correlated with decreased the phosphorylation expressions of Akt and FoxO3a. Moreover, high glucose incubation increased the expression of Bim, whereas increased anti-apoptotic protein BclxL. Furthermore, siRNA target FoxO3a silencing enhanced the ROS accumulation, whereas suppressed apoptosis in CMECs. FoxO3a silencing also abolished the disturbance of Bcl-2 proteins induced by high glucose in CMECs.
Conclusion
Our data provide evidence that high glucose induced FoxO3a activation which suppressed ROS accumulation, and in parallel, resulted in apoptosis of CMECs.
doi:10.1371/journal.pone.0079739
PMCID: PMC3832590  PMID: 24260294
5.  Novel compound heterozygous mutations in the MYO15A gene in autosomal recessive hearing loss identified by whole-exome sequencing 
Background
Inherited genetic defects play an important role in congenital hearing loss, contributing to about 60% of deafness occurring in infants. Hereditary nonsyndromic hearing loss is highly heterogeneous, and most patients with a presumed genetic etiology lack a specific molecular diagnosis.
Methods
By whole exome sequencing, we identified responsible gene of family 4794 with autosomal recessively nonsyndromic hearing loss (ARNSHL). We also used DNA from 56 Chinese familial patients with ARNSHL (autosomal recessive nonsyndromic hearing loss) and 108 ethnicity-matched negative samples to perform extended variants analysis.
Results
We identified MYO15A c.IVS25 + 3G > A and c.8375 T > C (p.V2792A) as the disease-causing mutations. Both mutations co-segregated with hearing loss in family 4794, but were absent in the 56 index patients and 108 ethnicity-matched controls.
Conclusions
Our results demonstrated that the hearing loss of family 4794 was caused by novel compound heterozygous mutations in MYO15A.
doi:10.1186/1479-5876-11-284
PMCID: PMC3828584  PMID: 24206587
Autosomal recessive sensorineural hearing loss; Whole-exome sequencing; MYO15A
6.  Assembly of Asperlicin Peptidyl Alkaloids from Anthranilate and Tryptophan: A Two-Enzyme Pathway Generates Heptacyclic Scaffold Complexity in Asperlicin E 
Journal of the American Chemical Society  2012;134(42):17444-17447.
Members of the asperlicin family of fungal metabolites produced by Aspergillus alliaceus are known potent CCKA antagonists. Herein we report the identification of the gene cluster responsible for directing their biosynthesis. We validate and probe the pathway by genetic manipulation, and provide the first biochemical characterization of the oxidative cyclization en route to the heptacyclic asperlicin E by reconstituting the activity of the FAD depend monooxygenase AspB. This report provides the first genetic characterization of a NRPS assembly line that efficiently activates two anthranilate building blocks and illustrates the remarkably efficient biosynthesis of the complex heptacyclic asperlicin E.
doi:10.1021/ja308371z
PMCID: PMC3500603  PMID: 23030663
7.  Primary angiosarcoma of the small intestine with metastasis to the liver: a case report and review of the literature 
Angiosarcoma is a rare disease with a poor prognosis; significantly, patients with intestinal angiosarcomas who survive over 1 year after diagnosis are extraordinarily rare. This article describes the case of a 33-year-old gentleman who presented with abdominal pain of 4 months duration, which had increased in severity 2 weeks prior to presentation. After a complicated diagnostic and therapeutic process, the diagnosis of primary angiosarcoma of the small intestine with metastasis to the liver was made by pathological and immunohistochemical examinations. We reviewed previous cases of angiosarcoma described in the English literature to determine their risk factors, diagnosis and treatment, and we found that angiosarcoma is extremely rare, especially in the small intestine. To the best of our knowledge, this may be the youngest case of primary angiosarcoma of the small intestine with metastasis to the liver reported in the English literature.
doi:10.1186/1477-7819-11-242
PMCID: PMC3851255  PMID: 24067058
Small intestine; Primary angiosarcoma; Hepatic metastasis
8.  Complexity Generation during Natural Product Biosynthesis using Redox Enzymes 
Current opinion in chemical biology  2012;16(3-4):362-369.
Redox enzymes such as FAD-dependent and cytochrome P450 oxygenases play indispensible roles in generating structural complexity during natural product biosynthesis. In the pre-assembly steps, redox enzymes can convert garden variety primary metabolites into unique starter and extender building blocks. In the post-assembly tailoring steps, redox cascades can transform nascent scaffolds into structurally complex final products. In this review, we will discuss several recently characterized redox enzymes in the biosynthesis of polyketides and nonribosomal peptides.
doi:10.1016/j.cbpa.2012.04.008
PMCID: PMC3415589  PMID: 22564679
9.  Tectorigenin Attenuates Palmitate-Induced Endothelial Insulin Resistance via Targeting ROS-Associated Inflammation and IRS-1 Pathway 
PLoS ONE  2013;8(6):e66417.
Tectorigenin is a plant isoflavonoid originally isolated from the dried flower of Pueraria thomsonii Benth. Although its anti-inflammatory and anti-hyperglycosemia effects have been well documented, the effect of tectorigenin on endothelial dysfunction insulin resistance involved has not yet been reported. Herein, this study aims to investigate the action of tectorigenin on amelioration of insulin resistance in the endothelium. Palmitic acid (PA) was chosen as a stimulant to induce ROS production in endothelial cells and successfully established insulin resistance evidenced by the specific impairment of insulin PI3K signaling. Tectorigenin effectively inhibited the ability of PA to induce the production of reactive oxygen species and collapse of mitochondrial membrane potential. Moreover, tectorigenin presented strong inhibition effect on ROS-associated inflammation, as TNF-α and IL-6 production in endothelial cells was greatly reduced with suppression of IKKβ/NF-κB phosphorylation and JNK activation. Tectorigenin also can inhibit inflammation-stimulated IRS-1 serine phosphorylation and restore the impaired insulin PI3K signaling, leading to a decreased NO production. These results demonstrated its positive regulation of insulin action in the endothelium. Meanwhile, tectorigenin down-regulated endothelin-1 and vascular cell adhesion molecule-1 overexpression, and restored the loss of insulin-mediated vasodilation in rat aorta. These findings suggested that tectorigenin could inhibit ROS-associated inflammation and ameliorated endothelial dysfunction implicated in insulin resistance through regulating IRS-1 function. Tectorigenin might have potential to be applied for the management of cardiovascular diseases involved in diabetes and insulin resistance.
doi:10.1371/journal.pone.0066417
PMCID: PMC3686685  PMID: 23840461
10.  Novel Compound Heterozygous TMC1 Mutations Associated with Autosomal Recessive Hearing Loss in a Chinese Family 
PLoS ONE  2013;8(5):e63026.
Hereditary nonsyndromic hearing loss is highly heterogeneous and most patients with a presumed genetic etiology lack a specific diagnosis. It has been estimated that several hundred genes may be associated with this sensory deficit in humans. Here, we identified compound heterozygous mutations in the TMC1 gene as the cause of recessively inherited sensorineural hearing loss by using whole-exome sequencing in a family with two deaf siblings. Sanger sequencing confirmed that both siblings inherited a missense mutation, c.589G>A p.G197R (maternal allele), and a nonsense mutation, c.1171C>T p.Q391X (paternal allele), in TMC1. We also used DNA from 50 Chinese familial patients with ARNSHL and 208 ethnicity-matched negative samples to perform extended variants analysis. Both variants co-segregated in family 1953, which had the hearing loss phenotype, but were absent in 50 patients and 208 ethnicity-matched controls. Therefore, we concluded that the hearing loss in this family was caused by novel compound heterozygous mutations in TMC1.
doi:10.1371/journal.pone.0063026
PMCID: PMC3653921  PMID: 23690975
11.  Cyclization of Fungal Nonribosomal Peptides by a Terminal Condensation-Like Domain 
Nature chemical biology  2012;8(10):823-830.
Cyclization of linear peptidyl precursors produced by nonribosomal peptide synthetases (NRPSs) is an important step in the biosynthesis of bioactive cyclic peptides. Whereas bacterial NRPSs use thioesterase (TE) domains to perform the cyclization, fungal NRPSs have apparently evolved to use a different enzymatic route. In verified fungal NRPSs that produce macrocyclic peptides, each megasynthetase terminates with a condensation-like (CT) domain that may perform the macrocyclization reaction. To probe the role of such a CT domain, we reconstituted the activities of the Penicillium aethiopicum trimodular NPRS TqaA in Saccharomyces cerevisiae and in vitro. Together with a reconstituted bimodular NRPS AnaPS, we dissected the cyclization steps of TqaA in transforming the linear anthranilate-D-tryptophan-L-alanyl tripeptide into fumiquinazoline F. Extensive biochemical and mutational studies confirmed the essential role of the CT domain in catalyzing cyclization in a thiolation domain-dependent fashion. Our work provided evidence of a likely universal macrocyclization strategy employed by fungal NRPSs.
doi:10.1038/nchembio.1047
PMCID: PMC3505271  PMID: 22902615
12.  Complexity Generation in Fungal Peptidyl Alkaloid Biosynthesis: Oxidation of Fumiquinazoline A to the Heptacyclic Hemiaminal Fumiquinazoline C by the Flavoenzyme Af12070 from Aspergillus fumigatus† 
Biochemistry  2011;50(40):8756-8769.
The human pathogen Aspergillus fumigatus makes a series of fumiquinazoline (FQ) peptidyl alkaloids of increasing scaffold complexity using L-Trp, two equivalents of L-Ala, and the non-proteinogenic amino acid anthranilate as building blocks. The FQ gene cluster encodes two nonribosomal peptide synthetases (NRPS) and two flavoproteins. The trimodular NRPS Af12080 assembles FQF (the first level of complexity) while the next two enzymes, Af12060 and Af12050, act in tandem in an oxidative annulation sequence to couple alanine to the indole side chain of FQF to yield the imidazolindolone-containing FQA. In this study we show that the fourth enzyme, the mono-covalent flavoprotein Af12070, introduces a third layer of scaffold complexity by converting FQA to the spirohemiaminal FQC, presumably by catalyzing the formation of a transient imine within the pyrazinone ring (and therefore acting in an unprecedented manner as an FAD-dependent amide oxidase). FQC subsequently converts non-enzymatically to the known cyclic aminal FQD. We also investigated the effect of substrate structure on Af12070 activity and subsequent cyclization with a variety of FQA analogues, including an FQA diastereomer (2′-epi-FQA) which is an intermediate in the fungal biosynthesis of the tremorgenic tryptoquialanine. 2′-epi-FQA is processed by Af12070 to epi-FQD, not epi-FQC, illustrating that the delicate balance in product cyclization regiochemistry can be perturbed by a remote stereochemical center.
doi:10.1021/bi201302w
PMCID: PMC3194008  PMID: 21899262
13.  Production of copolyesters of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by E. coli containing an optimized PHA synthase gene 
Background
Microbial polyhydroxyalkanoates (PHA) are biopolyesters consisting of diverse monomers. PHA synthase PhaC2Ps cloned from Pseudomonas stutzeri 1317 is able to polymerize short-chain-length (scl) 3-hydroxybutyrate (3HB) monomers and medium-chain-length (mcl) 3-hydroxyalkanoates (3HA) with carbon chain lengths ranging from C6 to C12. However, the scl and mcl PHA production in Escherichia coli expressing PhaC2Ps is limited with very low PHA yield.
Results
To improve the production of PHA with a wide range of monomer compositions in E. coli, a series of optimization strategies were applied on the PHA synthase PhaC2Ps. Codon optimization of the gene and mRNA stabilization with a hairpin structure were conducted and the function of the optimized PHA synthase was tested in E. coli. The transcript was more stable after the hairpin structure was introduced, and western blot analysis showed that both codon optimization and hairpin introduction increased the protein expression level. Compared with the wild type PhaC2Ps, the optimized PhaC2Ps increased poly-3-hydroxybutyrate (PHB) production by approximately 16-fold to 30% of the cell dry weight. When grown on dodecanoate, the recombinant E. coli harboring the optimized gene phaC2PsO with a hairpin structure in the 5’ untranslated region was able to synthesize 4-fold more PHA consisting of 3HB and medium-chain-length 3HA compared to the recombinant harboring the wild type phaC2Ps.
Conclusions
The levels of both PHB and scl-mcl PHA in E. coli were significantly increased by series of optimization strategies applied on PHA synthase PhaC2Ps. These results indicate that strategies including codon optimization and mRNA stabilization are useful for heterologous PHA synthase expression and therefore enhance PHA production.
doi:10.1186/1475-2859-11-130
PMCID: PMC3503839  PMID: 22978778
PHB; Polyhydroxyalkanoates; PHA synthase; Codon optimization; Hairpin; Escherichia coli
14.  Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice 
Carcinogenesis  2011;32(9):1396-1402.
M3 subtype muscarinic receptors (CHRM3) are over-expressed in colon cancer. In this study, we used Apcmin/+ mice to identify the role of Chrm3 expression in a genetic model of intestinal neoplasia, explored the role of Chrm3 in intestinal mucosal development and determined the translational potential of inhibiting muscarinic receptor activation. We generated Chrm3-deficient Apcmin/+ mice and compared intestinal morphology and tumor number in 12-week-old Apcmin/+Chrm3−/− and Apcmin/+Chrm3+/+ control mice. Compared with Apcmin/+Chrm3+/+ mice, Apcmin/+Chrm3−/− mice showed a 70 and 81% reduction in tumor number and volume, respectively (P < 0.01). In adenomas, β-catenin nuclear staining was reduced in Apcmin/+Chrm3−/− compared with Apcmin/+Chrm3+/+ mice (P < 0.02). Whereas Apc gene mutation increased the number of crypt and Paneth cells and decreased villus goblet cells, these changes were absent in Apcmin/+Chrm3−/− mice. To determine whether pharmacological inhibition of muscarinic receptor activation attenuates intestinal neoplasia, we treated 6-week-old Apcmin/+ mice with scopolamine butylbromide, a non-subtype-selective muscarinic receptor antagonist. After 8 weeks of continuous treatment, scopolamine butylbromide-treated mice showed a 22% reduction in tumor number (P = 0.027) and a 36% reduction in tumor volume (P = 0.004) as compared with control mice. Compared with Chrm3 gene ablation, the muscarinic antagonist was less efficacious, most probably due to shorter duration of treatment and incomplete blockade of muscarinic receptors. Overall, these findings indicate that interplay of Chrm3 and β-catenin signaling is important for intestinal mucosal differentiation and neoplasia and provide a proof-of-concept that pharmacological inhibition of muscarinic receptor activation can attenuate intestinal neoplasia in vivo.
doi:10.1093/carcin/bgr118
PMCID: PMC3165126  PMID: 21705482
15.  Adolescent Bullying Involvement and Psychosocial Aspects of Family and School Life: A Cross-Sectional Study from Guangdong Province in China 
PLoS ONE  2012;7(7):e38619.
Background
School bullying is an emerging problem in China. The present study aimed to measure the prevalence of bullying behaviors among Chinese adolescents and to examine the association of bullying and being bullied with family factors, school factors and indicators of psychosocial adjustment.
Methods
A cross-sectional study was conducted. A total of 8,342 middle school students were surveyed in four cities in the Guangdong Province. Self-reports on bullying involvement and information regarding family factors, school factors and psychosocial adjustment were collected. Descriptive statistics and multi-level logistic regression analysis were used to evaluate the prevalence of school bullying and explore potentially influential factors.
Results
Of the total sample, 20.83% (1,738) reported being involved in bullying behaviors. Of the respondents, 18.99% were victims of bullying, 8.60% were bullies and 6.74% both bullied themselves and bullied others. Factors that were determined to be correlated with bullying behaviors included grade, parental caring, consideration of suicide, running away from home, time spent online per day and being in a physical fight.
Conclusion
Bullying was determined to be prevalent among Chinese adolescents. Given the concurrent psychosocial adjustment, family and school factors associated with bullying, as well as the potential long-term negative outcomes for these youth, this issue merits serious attention, both for future research and preventive intervention.
doi:10.1371/journal.pone.0038619
PMCID: PMC3399853  PMID: 22815693
16.  Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites† 
Biochemistry  2011;50(25):5668-5679.
The fungal peptidyl alkaloids of the tryptoquialanine and fumiquinazoline families are nonribosomally assembled by annulation of the indole side chain of fumiquinazoloine F (FQF) with an alaninyl or aminoisobutyryl unit by monomodular NRPS enzymes containing adenylation, thiolation and condensation (A-T-C) domains. The enzyme pair Af12060 and Af12050 from Aspergillus fumigatus thereby convert FQF to FQA while the homologous enzyme pair TqaH and TqaB from Penicillium aethiopicum make the 2′-epi diastereomer of FQA, differing only in the stereochemistry of one of the C-N bonds formed in the annulation with L-Ala. To evaluate the basis for this stereochemical control we have mixed and matched the flavoprotein oxygenases Af12060 and TqaH with the A-T-C modular enzymes Af12050 and TqaB to show that the NRPS enzymes control the stereochemical outcome. The terminal 50 kDa condensation domains of Af12050 and TqaB are solely responsible for the stereochemical control as shown both by making chimeric (e.g. A-T-C* and A*-T*-C ) forms of these monomodular NRPS enzymes and also by expression, purification and assay of the excised C domains. The Af12050 and TqaB condensation domains are thus a paired set of diastereospecific annulation catalysts that act on the fumiquinazoline F scaffold.
doi:10.1021/bi2004922
PMCID: PMC3119741  PMID: 21591693
17.  Gastric mucosal damage in water immersion stress: Mechanism and prevention with GHRP-6 
AIM: To investigate the mechanism of gastric mucosal demage induced by water immersion restraint stress (WRS) and its prevention by growth hormone releasing peptide-6 (GHRP-6).
METHODS: Male Wistar rats were subjected to conscious or unconscious (anesthetized) WRS, simple restraint (SR), free swimming (FS), non-water fluid immersion, immersion without water contact, or rats were placed in a cage surrounded by sand. To explore the sensitivity structures that influence the stress reaction besides skin stimuli, a group the rats had their eyes occluded. Cervical bilateral trunk vagotomy or atropine injection was performed in some rats to assess the parasympathetic role in mucosal damage. Gastric mucosal lesions, acid output and heart rate variability were measured. Plasma renin, endothelin-1 and thromboxane B2 and gastric heat shock protein 70 were also assayed. GHRP-6 was injected [intraperitoneal (IP) or intracerebroventricular (ICV)] 2 h before the onset of stress to observe its potential prevention of the mucosal lesion.
RESULTS: WRS for 6 h induced serious gastric mucosal lesion [lesion area, WRS 81.8 ± 6.4 mm2 vs normal control 0.0 ± 0.0 mm2, P < 0.01], decreased the heart rate, and increased the heart rate variability and gastric acid secretion, suggesting an increase in vagal nerve-carrying stimuli. The mucosal injury was inversely correlated with water temperature (lesion area, WRS at 35 °C 56.4 ± 5.2 mm2 vs WRS at 23 °C 81.8 ± 6.4 mm2, P < 0.01) and was consciousness-dependent. The injury could not be prevented by eye occlusion, but could be prevented by avoiding contact of the rat body with the water by dressing it in an impermeable plastic suit. When water was replaced by vegetable oil or liquid paraffin, there were gastric lesions in the same grade of water immersion. When rat were placed in a cage surrounded by sand, there were no gastric lesions. All these data point to a remarkable importance of cutenuous information transmitted to the high neural center that by vagal nerves reaching the gastric mucosa. FS alone also induced serious gastric injury, but SR could not induce gastric injury. Bilateral vagotomy or atropine prevented the WRS-induced mucosal lesion, indicating that increased outflow from the vagal center is a decisive factor in WRS-induced gastric injury. The mucosal lesions were prevented by prior injection of GHRP-6 via IP did, but not via ICV, suggesting that the protection is peripheral, although a sudden injection is not equivalent to a physiological release and uptake, which eventually may affect the vagal center.
CONCLUSION: From the central nervous system, vagal nerves carry the cutaneous stimuli brought about by the immersion restraint, an experimental model for inducing acute gastric erosions. GHRP-6 prevents the occurrence of these lesions.
doi:10.3748/wjg.v18.i24.3145
PMCID: PMC3386329  PMID: 22791951
Growth substances; Gastric ulcer; Stress; Behavior and emotions; Autonomic nerve; Heart rate variability
18.  Priority in Selenium Homeostasis Involves Regulation of SepSecS Transcription in the Chicken Brain 
PLoS ONE  2012;7(4):e35761.
O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) is critical for the biosynthesis and transformation of selenocysteine (Sec) and plays an important role in the biological function of Se through the regulation of selenoprotein synthesis. Selenium (Se) and Selenoprotein play a pivotal role in brain function. However, how intake of the micronutrient Se affects gene expression and how genetic factors influence Se metabolism in the brain is unknown. To investigate the regulation of SepSecS transcription induced by Se in the chicken brain, we determined the Se content of brain tissue, SepSecS gene expression levels and mRNA stability in the chicken brain and primary cultured chicken embryos neurons receiving Se supplements. These results showed that Se content in the brain remains remarkably stable during Se supplementation. A significant increase in SepSecS mRNA levels was observed in all of the brain tissues of chickens fed diets containing 1–5 mg/kg sodium selenite. Most strikingly, significant changes in SepSecS mRNA levels were not observed in neurons treated with Se. However, Se altered the SepSecS mRNA half-life in cells. These data suggest that Se could regulate SepSecS mRNA stability in the avian brain and that SepSecS plays an important role in Se homeostasis regulation.
doi:10.1371/journal.pone.0035761
PMCID: PMC3334997  PMID: 22536434
19.  5α-Hy­droxy­eudesm-4(15),11(13)-dien-8β,12-olide 
The title compound, C15H20O3, a sesquiterpene lactone, was isolated from the aerial parts of Carpesium minus Hemsl. (Compositae). The mol­ecule is composed of three rings, with the two cyclo­hexane rings in chair conformations and the cyclo­pentane ring adopting a twist conformation. The A/B ring junction is trans-fused. The absolute configuration shown has been arbitrarily assigned. In the crystal, mol­ecules are linked into [100] chains by O—H⋯O hydrogen bonds.
doi:10.1107/S1600536812012470
PMCID: PMC3344144  PMID: 22606147
20.  Fungal Indole Alkaloid Biosynthesis: Genetic and Biochemical Investigation of Tryptoquialanine Pathway in Penicillium aethiopicum 
Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline 2 that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6-5-5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P. aethiopicum that is involved in the biosynthesis of tryptoquialanine 1, which is structurally similar to 2. The pathway has been confirmed to go through an intermediate common to the fumiquinazoline pathway, fumiquinazoline F, which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). By systematically inactivating every biosynthetic gene in the cluster, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of the pathway. An unusual oxidative opening of the pyrazinone ring by an FAD-dependent berberine bridge enzyme-like oxidoreductase has been proposed based on genetic knockout studies. Notably, a 2-aminoisobutyric acid (AIB)-utilizing NRPS module has been identified and reconstituted in vitro, along with two putative enzymes of unknown functions that are involved in the synthesis of the unnatural amino acid by genetic analysis. This work provides new genetic and biochemical insights into the biosynthesis of this group of fungal alkaloids, including the tremorgens related to 2.
doi:10.1021/ja1101085
PMCID: PMC3045477  PMID: 21299212
mycotoxins; alkaloids; aminoisobutyrate; oxidoreductase
21.  catena-Poly[[(1,10-phenanthroline)cobalt(II)]-di-μ-azido] 
In the crystal structure of the binuclear title complex, [Co(N3)2(C12H8N2)]n, each CoII cation is coordinated by two N atoms from one chelating 1,10-phenanthroline ligand and four azide ligands in a slightly distorted octa­hedral coordination. The two CoII cations of the binuclear complex are related by an inversion centre and are bridged by two symmetry-related azide ligands in both μ1,1 and μ1,3 modes. The μ1,3 bridging mode gives rise to an infinite one-dimensional chain along the a axis, whereas the μ1,1 bridging mode is responsible for the formation of the binuclear CoII complex.
doi:10.1107/S1600536812006435
PMCID: PMC3297267  PMID: 22412457
22.  Expression, purification and characterization of Mycobacterium tuberculosis RpfE protein☆ 
Journal of Biomedical Research  2012;26(1):17-23.
Resuscitation promoting factor E (RpfE) is one of the five Rpf-like proteins in Mycobacterium tuberculosis (M. tuberculosis). These Rpf-like proteins are secretory, which make them candidates for recognition by the host immune system. In this study, the RpfE gene was amplified from M. tuberculosis, cloned into the expression vectors pDE22 and pPRO EXHT, and were expressed in Mycobacterium vaccae (M. vaccae) and Escherichia coli DH5α, respectively. Both recombinant RpfE proteins were purified by Ni-Sepharose affinity chromatography, and were given to C57BL/6 mice. The RpfE proteins elicited T cell proliferation, and stimulated the production of gamma interferon (IFN-γ), interleukin-10 (IL-10) and IL-12. Our results indicated that the RpfE protein expressed in M. vaccae could more efficiently stimulate cellular immune response, making it a promising candidate as a subunit vaccine.
doi:10.1016/S1674-8301(12)60003-7
PMCID: PMC3596076  PMID: 23554726
resuscitation-promoting factor (RpfE); purification; Mycobacterium tuberculosis; Mycobacterium vaccae
23.  Co-Chaperone HSJ1a Dually Regulates the Proteasomal Degradation of Ataxin-3 
PLoS ONE  2011;6(5):e19763.
Homo sapiens J domain protein (HSJ1) is a J-domain containing co-chaperone that is known to stimulate ATPase activity of HSP70 chaperone, while it also harbors two ubiquitin (Ub)-interacting motifs (UIMs) that may bind with ubiquitinated substrates and potentially function in protein degradation. We studied the effects of HSJ1a on the protein levels of both normal and the disease–related polyQ-expanded forms of ataxin-3 (Atx3) in cells. The results demonstrate that the N-terminal J-domain and the C-terminal UIM domain of HSJ1a exert opposite functions in regulating the protein level of cellular overexpressed Atx3. This dual regulation is dependent on the binding of the J-domain with HSP70, and the UIM domain with polyUb chains. The J-domain down-regulates the protein level of Atx3 through HSP70 mediated proteasomal degradation, while the UIM domain may alleviate this process via maintaining the ubiquitinated Atx3. We propose that co-chaperone HSJ1a orchestrates the balance of substrates in stressed cells in a Yin-Yang manner.
doi:10.1371/journal.pone.0019763
PMCID: PMC3098244  PMID: 21625540
24.  The neutrophil/lymphocyte ratio is an independent prognostic indicator in patients with bone metastasis 
Oncology Letters  2011;2(4):735-740.
Patients with the most common advanced human cancers such as lung, breast, uterus, and cancers of the digestive system almost always develop bone metastases, with painful and untreatable consequences. This study aimed to determine the prognostic implications of the neutrophil/lymphocyte (N/L) ratio in the peripheral blood of patients with malignant bone metastasis. Study participants were identified from a prospective cohort of cancer patients with bone metastasis. Data for the N/L ratios were obtained from clinical and pathological records and were analyzed together with other known prognostic factors in the multivariate and univariate analyses. The results showed the average N/L ratio of all 497 patients to be 4.25±2.44 (range 0.54–45.50 years). Multivariate analysis revealed that tumor type and a high N/L ratio were significantly associated with poor prognosis. For the high N/L ratio group, the estimated hazard ratio of death was 1.348 [95% confidence interval (CI), 1.062–1.712] compared with the low N/L ratio group. The average N/L ratio of the 225 patients in the surgery group was 2.79±2.46 (range 0.77–22.75 years). Multivariate analysis revealed that a preoperatively high N/L ratio (P=0.013; HR=2.945; 95% CI, 1.256–6.906) was significantly associated with poor prognosis after bone metastasis in the surgery group. In conclusion, the N/L ratio was confirmed to be an independent prognostic factor in patients with bone metastasis. Thus, the N/L ratio may serve as a clinically accessible and useful biomarker for patient survival.
doi:10.3892/ol.2011.304
PMCID: PMC3406337  PMID: 22848258
bone metastasis; neutrophil/lymphocyte ratio; prognostic indicator
25.  Endothelium-derived hyperpolarizing factor and diabetes 
World Journal of Cardiology  2011;3(1):25-31.
In addition to its role as a barrier between blood and tissues, the vascular endothelium is responsible for the synthesis and released of a number of vasodilators including prostaglandins, nitric oxide and endothelium-derived hyperpolarizing factor (EDHF). As one of these vasodilators, the specific nature of EDHF has not been fully elucidated, although a number of roles have been proposed. Importantly, many conditions, such as hypertension, hyperlipidemia, heart failure, ischemia-reperfusion and diabetes mellitus comprise vascular endothelial dysfunction with EDHF dysregulation. This article reviews reports on the role of EDHF in diabetes-related endothelial dysfunction.
doi:10.4330/wjc.v3.i1.25
PMCID: PMC3030734  PMID: 21286215
Endothelial-dysfunction; Hyperglycemia; Potassium channels; Epoxyeicosatrienoic acids; Hydrogen peroxide

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