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1.  BRAF Mutations in Patients with Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(6):e101354.
Background
BRAF mutations have been well described in non-small cell lung cancer (NSCLC) for several years, but the clinical features of patients harboring BRAF mutations are still not well described. We performed a meta-analysis to identify common clinical features in NSCLC patients carrying BRAF mutations.
Methods
We identified clinical studies that examined the association between BRAF mutations and features of NSCLC within PubMed, Embase and ISI Science Citation Index database up to October 2013. The effect size of clinical features was estimated by odds ratios (ORs) with 95% confidence interval (CI) for each study, using a fixed-effects or random-effects model.
Results
Ten studies with a total of 5599 NSCLC patients were included. There was a 3% (170/5599) BRAF mutation rate. BRAF mutations in NSCLC were significantly associated with adenocarcinomas (ADCs) (compared with non-ADCs, OR = 4.96, 95%CI = 2.29–10.75). There were no significant differences in gender, smoking and stage in patients with and without BRAF mutations. The BRAFV600E mutation was more frequent in women than non-BRAFV600E mutations (OR = 0.27, 95%CI = 0.12–0.59), and was closely related to never smokers (OR = 0.14, 95%CI = 0.05–0.42).
Conclusions
These findings have important implications for the prediction of the NSCLC sub-types more accurately combined with other genetic changes.
doi:10.1371/journal.pone.0101354
PMCID: PMC4076330  PMID: 24979348
2.  Visualizing GroEL/ES in the Act of Encapsulating a Folding Protein 
Cell  2013;153(6):1354-1365.
Summary
The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle electron cryo-microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involves concerted shifts of GroES and the GroEL apical domains and C-terminal tails.
doi:10.1016/j.cell.2013.04.052
PMCID: PMC3695626  PMID: 23746846
3.  Polyethylene Glycol Electrolyte Lavage Solution versus Colonic Hydrotherapy for Bowel Preparation before Colonoscopy: A Single Center, Randomized, and Controlled Study 
This single center, randomized, and controlled study aimed to compare the effectiveness and safety of polyethylene glycol electrolyte lavage (PEG-EL) solution and colonic hydrotherapy (CHT) for bowel preparation before colonoscopy. A total of 196 eligible outpatients scheduled for diagnostic colonoscopy were randomly assigned to the PEG-EL (n = 102) or CHT (n = 94) groups. Primary outcome measures included colonic cleanliness and adverse effects. Secondary outcome measures were patient satisfaction and preference, colonoscopic findings, ileocecal arrival rate, examiner satisfaction, and cecal intubation time. The results show that PEG-EL group was associated with significantly better colonic cleanliness than CHT group, fewer adverse effects, and increased examiner satisfaction. However, the CHT group had higher patient satisfaction and higher diverticulosis detection rates. Moreover, the results showed the same ileocecal arrival rate and patient preference between the two groups (P > 0.05). These findings indicate that PEG-EL is the preferred option in patients who followed the preparation instructions completely.
doi:10.1155/2014/541586
PMCID: PMC4068103  PMID: 24995014
4.  Direct Ethanol Production from Lignocellulosic Sugars and Sugarcane Bagasse by a Recombinant Trichoderma reesei Strain HJ48 
The Scientific World Journal  2014;2014:798683.
Trichoderma reesei can be considered as a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here the ethanol production of T. reesei CICC 40360 was improved by genome shuffling while simultaneously enhancing the ethanol resistance. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing more than fivefold ethanol than wild type was obtained by genome shuffling. The results show that the shuffled strain HJ48 can efficiently convert lignocellulosic sugars to ethanol under aerobic conditions. Furthermore, it was able to produce ethanol directly from sugarcane bagasse, demonstrating that the shuffled strain HJ48 is a suitable microorganism for consolidated bioprocessing.
doi:10.1155/2014/798683
PMCID: PMC4060538  PMID: 24995362
5.  Preadaptation to Cold Stress in Salmonella enterica Serovar Typhimurium Increases Survival during Subsequent Acid Stress Exposure 
Applied and Environmental Microbiology  2013;79(23):7281-7289.
Salmonella is an important cause of bacterial food-borne gastroenteritis. Salmonella encounters multiple abiotic stresses during pathogen elimination methods used in food processing, and these stresses may influence its subsequent survivability within the host or in the environment. Upon ingestion, Salmonella is exposed to gastrointestinal acidity, a first line of the host innate defense system. This study tested the hypothesis that abiotic stresses encountered during food processing alter the metabolic mechanisms in Salmonella that enable survival and persistence during subsequent exposure to the host gastrointestinal acidic environment. Out of the four different abiotic stresses tested, viz., cold, peroxide, osmotic, and acid, preadaptation of the log-phase culture to cold stress (5°C for 5 h) significantly enhanced survival during subsequent acid stress (pH 4.0 for 90 min). The gene expression profile of Salmonella preadapted to cold stress revealed induction of multiple genes associated with amino acid metabolism, oxidative stress, and DNA repair, while only a few of the genes in the above-mentioned stress response and repair pathways were induced upon exposure to acid stress alone. Preadaptation to cold stress decreased the NAD+/NADH ratio and hydroxyl (OH·) radical formation compared with those achieved with the exposure to acid stress alone, indicating alteration of aerobic respiration and the oxidative state of the bacteria. The results from this study suggest that preadaptation to cold stress rescues Salmonella from the deleterious effect of subsequent acid stress exposure by induction of genes involved in stress response and repair pathways, by modification of aerobic respiration, and by redox modulation.
doi:10.1128/AEM.02621-13
PMCID: PMC3837722  PMID: 24056458
6.  Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings 
Proteome Science  2014;12:31.
Background
Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms.
Methods
Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry.
Results
A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases.
Conclusions
Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations.
doi:10.1186/1477-5956-12-31
PMCID: PMC4046019  PMID: 24904238
Apple; Vegetative phase change; Floral transition; Phosphorylated proteomics
7.  In vivo determination of muscle-derived stem cells in the rat corpus cavernosum 
The aim of the present in vivo study was to determine the presence of muscle-derived stem cells (MDSCs) in the corpus cavernosum of rats. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were performed to detect the expression of the stem cell markers stem cell antigen-1 (Sca-1), Oct4 and Desmin in Sprague-Dawley rats aged 2, 5 and 20 months. Sca-1 was mainly expressed in the blood vessels and cavernous sinus and staining revealed that Sca-1 was predominantly expressed in the cytoplasm. Desmin was primarily expressed in muscular tissues and staining demonstrated that it was mainly expressed in the cytoplasm, however, Desmin was also partially expressed in the nuclei. A small number of double positive cells, expressing Sca-1 and Desmin, were also detected near the cavernous sinus. It was found that the expression of the markers was negatively correlated with the age of the rats (P<0.05). The results from the RT-PCR demonstrated that the expression levels of Sca-1 and Desmin significantly decreased with age (P<0.05). In addition, the correlation analysis indicated that the expression of Sca-1 and Desmin were negatively correlated with the age of the rats (r=−−0.929; P<0.05). In conclusion, the present study provided evidence for the presence of MDSCs in the rat corpus cavernosum. MDSCs may be a potential therapeutic treatment for organic erectile dysfunction.
doi:10.3892/etm.2014.1710
PMCID: PMC4061231  PMID: 24944634
muscle-derived stem cells; rat corpus cavernosum; stem cell markers; erectile dysfunction
8.  Nitric Oxide and Carbon Monoxide Production and Metabolism in Preeclampsia 
Reproductive Sciences  2013;20(5):542-548.
Objective:
To elucidate the regulation of the nitric oxide (NO) and carbon monoxide (CO) pathways in preeclampsia and to evaluate the ratio of asymmetric dimethylarginine (ADMA) to symmetric dimethylarginine (SDMA) as a marker for preeclampsia.
Methods:
Maternal plasma and placental samples were obtained from 20 participants with preeclampsia and 23 controls. Enzyme-linked immunosorbent assay was used to measure plasma NO, ADMA, and SDMA as well as placental NO and hemeoxygnase 1 (HO-1). Western blot was used to measure placental dimethylarginine dimethylaminotransferases (DDAH-I and DDAH-II).
Results:
Placental DDAH-I, placental DDAH-II, placental NO, and placental HO-1 were significantly decreased in participants with preeclampsia. While ADMA and SDMA levels were decreased in preeclampsia, the ADMA-SDMA ratio was not significantly different.
Conclusions:
Decreased DDAH and HO with preeclampsia suggest that they are important points in the regulatory pathways of NO and CO production that are altered in preeclampsia. The ADMA-SDMA ratio is not a useful test for preeclampsia.
doi:10.1177/1933719112459231
PMCID: PMC3713541  PMID: 23012314
asymmetric dimethylarginine; carbon monoxide; hemeoxygenase; nitric oxide; preeclampsia
9.  Transcriptional and Functional Adaptations of Human Endothelial Cells to Physiological Chronic Low Oxygen1 
Biology of Reproduction  2013;88(5):114.
ABSTRACT
Endothelial cells chronically reside in low-O2 environments in vivo (2%–13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; 21% O2) or physiological chronic normoxia (PCN; 3% O2) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated 21 genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O2. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to 21% O2 up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.
Physiological chronic low oxygen alters transcription and function of human endothelial cells.
doi:10.1095/biolreprod.113.108225
PMCID: PMC4013893  PMID: 23536375
angiogenesis; growth factors; physiological chronic low oxygen; protein kinases; transcriptome
10.  Human Placental MicroRNAs and Preeclampsia1 
Biology of Reproduction  2013;88(5):130.
ABSTRACT
MicroRNAs are a class of noncoding small RNAs that regulate the expression of nearly 30% of all the human genes and participate in all fundamental cell processes. Genome-wide analysis has revealed that human placenta expresses more than 600 miRNA species, including placenta-specific ones with high levels of expression. Comparative analysis also has revealed many differentially expressed miRNAs with either high or low levels of expression in human placentas from normal versus preeclamptic pregnancies, indicating an important role of miRNAs in normal and pathological placental physiology. Although limited information is currently available as to how miRNA regulates human placental development and function, there are studies suggesting that preeclampsia-associated differentially expressed miRNAs possess critical roles in regulating placental development and function via targeting specific genes with diverse known functions. Herein we summarize the current findings regarding the expression of placental miRNAs and their function, especially in the trophoblast cells. We have recently found that the angiogenesis-associated miR-17-family miRNAs are upregulated in preeclamptic compared with normotensive placentas and they target the ephrin-B2/Eph receptor B4 (EPHB4) system. Because ephrin-B2 and EPHB4 has been previously shown to play a crucial role in trophoblast invasion into maternal spiral artery and vascular patterning during early human placental development, the miR-17-ephrin-B2/EPHB4 pathway seems to be a novel miRNA pathway for regulating normal and aberrant placental development during preeclampsia.
Small noncoding microRNAs have major roles in human placental development and the pathogenesis of preeclampsia.
doi:10.1095/biolreprod.113.107805
PMCID: PMC4013914  PMID: 23575145
microRNA; placenta; placentation; preeclampsia; pregnancy
11.  Microwave-assisted green synthesis of Ag/reduced graphene oxide nanocomposite as a surface-enhanced Raman scattering substrate with high uniformity 
Nanoscale Research Letters  2014;9(1):193.
A nanocomposite of silver nanoparticles/reduced graphene oxide (Ag/rGO) has been fabricated as a surface-enhanced Raman scattering (SERS) substrate owing to the large surface area and two-dimensional nanosheet structure of rGO. A facile and rapid microwave-assisted green route has been used for the formation of Ag nanoparticles and the reduction of graphene oxide simultaneously with L-arginine as the reducing agent. By increasing the cycle number of microwave irradiation from 1 and 4 to 8, the mean diameters of Ag nanoparticles deposited on the surface of rGO increased from 10.3 ± 4.6 and 21.4 ± 10.5 to 41.1 ± 12.6 nm. The SERS performance of Ag/rGO nanocomposite was examined using the common Raman reporter molecule 4-aminothiophenol (4-ATP). It was found that the Raman intensity of 4-ATP could be significantly enhanced by increasing the size and content of silver nanoparticles deposited on rGO. Although the Raman intensities of D-band and G-band of rGO were also enhanced simultaneously by the deposited Ag nanoparticles which limited the further improvement of SERS detection sensitivity, the detectable concentration of 4-ATP with Ag/rGO nanocomposite as the SERS substrate still could be lowered to be 10−10 M and the enhancement factor could be increased to 1.27 × 1010. Furthermore, it was also achievable to lower the relative standard deviation (RSD) values of the Raman intensities to below 5%. This revealed that the Ag/rGO nanocomposite obtained in this work could be used as a SERS substrate with high sensitivity and homogeneity.
doi:10.1186/1556-276X-9-193
PMCID: PMC4005903  PMID: 24808800
Reduced graphene oxide; Ag nanoparticles; Microwave; Green synthesis; SERS substrate; Uniformity
12.  Differential expressions of BMPR1α, ACTN4α and FABP7 in Hirschsprung disease 
Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. Recent studies have shown that the bone morphogenetic protein receptor-type IA (BMPR1α), actinin-alpha 4 (ACTN4α) and fatty acid binding protein 7 (FABP7) play important roles in the differentiation and development of neurons. The aganglionic (stenotic) and the ganglionic (normal) colon segment tissues of 60 HSCR patients were collected to investigate the expression pattern of BMPR1α, ACTININ-4α and FABP7 using RT-PCR, quantitative real-time RT-PCR (qRT-PCR) and immunohistochemical staining. The mRNA and protein expressions of BMPR1α and ACTN4α were higher in the stenotic colon segment tissue than those in the normal colon segment tissue. However, the mRNA and protein expressions of FABP7 were lower in the stenotic colon segment tissue than those in the normal colon segment tissue. The study in HSCR patients, findings in mRNA and protein alterations to expecting provide more information to in order to find some clue for the pathomechanism of HSCR disease.
PMCID: PMC4069943  PMID: 24966941
Hirschsprung disease (HSCR); BMPR1α; ACTININ-4α and FABP7; aganglionic (stenotic) colon segment; ganglionic (normal) colon segment
13.  Spatiotemporal expression of Wnt5a during the development of the striated muscle complex in rats with anorectal malformations 
Fecal incontinence and constipation after procedures for anorectal malformations (ARMs) are closely related to the maldevelopment of the striated muscle complex (SMC). Previous studies have demonstrated that myogenic regulatory factors (MRFs) play a significant role in muscle development. Wnt signal pathway is extremely important for MRFs regulation. This study was designed to investigate the spatiotemporal expression pattern of Wnt5a in SMC in ARMs rat embryos. Materials and Methods: Anorectal malformation embryos were induced by ethylene thiourea on embryonic day 10 (E10). Expression levels of protein and mRNA of Wnt5a were confirmed by immunohistochemistry staining, western blot and quantitative real-time PCR (qRT-PCR) between normal rat embryos and embryos with ARMs. Results: Immunostaining revealed that, on embryonic day 17 (E17), the Wnt5a protein was initially expressed in the SMC in normal embryos. With the growth of pregnancy, the positive staining cells gradually increased. The same time-dependent changes of Wnt5a protein were detected in ARMs embryos. Besides, immunostaining showed that Wnt5a had a significant increase in normal embryos compared with ARMs embryos. Similarly, in Western blot and qRT-PCR, the higher expression of Wnt5a protein and mRNA were remarkable in normal embryos during the SMC development, relatively. Conclusion: Our study demonstrated that the downregulation of Wnt5a at the time of SMC development might partly be related to the dysplasia of SMC in ARMs.
PMCID: PMC4069947  PMID: 24966909
Anorectal malformations; Wnt5a; striated muscle complex; expression
14.  In Depth Analysis on the Binding Sites of Adamantane Derivatives in HCV (Hepatitis C Virus) p7 Channel Based on the NMR Structure 
PLoS ONE  2014;9(4):e93613.
Background
The recently solved solution structure of HCV (hepatitis C virus) p7 ion channel provides a solid structure basis for drug design against HCV infection. In the p7 channel the ligand amantadine (or rimantadine) was determined in a hydrophobic pocket. However the pharmocophore (−NH2) of the ligand was not assigned a specific binding site.
Results
The possible binding sites for amino group of adamantane derivatives is studied based on the NMR structure of p7 channel using QM calculation and molecular modeling. In the hydrophobic cavity and nearby three possible binding sites are proposed: His17, Phe20, and Trp21. The ligand binding energies at the three binding sites are studied using high level QM method CCSD(T)/6–311+G(d,p) and AutoDock calculations, and the interaction details are analyzed. The potential application of the binding sites for rational inhibitor design are discussed.
Conclusions
Some useful viewpoints are concluded as follows. (1) The amino group (−NH2) of adamantane derivatives is protonated (−NH3+), and the positively charged cation may form cation-π interactions with aromatic amino acids. (2) The aromatic amino acids (His17, Phe20, and Trp21) are the possible binding sites for the protonated amino group (−NH3+) of adamantane derivatives, and the cation-π bond energies are 3 to 5 times stronger than the energies of common hydrogen bonds. (3) The higher inhibition potent of rimantadine than amantadine probably because of its higher pKa value (pKa = 10.40) and the higher positive charge in the amino group. The potential application of p7 channel structure for inhibitor design is discussed.
doi:10.1371/journal.pone.0093613
PMCID: PMC3979700  PMID: 24714586
15.  Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube 
PLoS ONE  2014;9(4):e91824.
Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques.
doi:10.1371/journal.pone.0091824
PMCID: PMC3976262  PMID: 24705376
16.  Cryo-EM Structure of a Molluscan Hemocyanin Suggests its Allosteric Mechanism 
Summary
Hemocyanins are responsible for transporting O2 in the arthropod and molluscan hemolymph. Haliotis diversicolor molluscan hemocyanin isoform 1 (HdH1) is an 8 MDa oligomer. Each subunit is made up of eight functional units (FUs). Each FU contains two Cu ions, which can reversibly bind an oxygen molecule. Here we report a ~4.5 Å cryo-EM structure of HdH1. The structure clearly shows 10 asymmetric units arranged with D5 symmetry. Each asymmetric unit contains two structurally distinct but chemically identical subunits. The map is sufficiently resolved to trace the entire subunit Cα backbone, and to visualize densities corresponding to some large sidechains, Cu ion pairs, and interaction networks of adjacent subunits. A FU topology path intertwining between the two subunits of the asymmetric unit is unambiguously determined. Our observations suggest a structural mechanism for the stability of the entire hemocyanin didecamer and 20 “communication clusters” across asymmetric units responsible for its allosteric property upon oxygen binding.
doi:10.1016/j.str.2013.02.018
PMCID: PMC3657317  PMID: 23541894
17.  WNT3A gene expression is associated with isolated Hirschsprung disease polymorphism and disease status 
WNT3A has been regarded as an activator of the canonical Wnt signaling pathway. It has been found Wnt signaling pathway is closely related with embrionic development and Hirschsprung disease (HSCR). A common haplotype consisting of minor SNPs alleles located in the WNT3A gene has been described as a risk factor for various genetic disorders. However, whether WNT3A contributes to the onset of HSCR has not been identified. The present study aims to detect the interactions of genetic variations in the WNT3A gene and examine the biological expression levels with Hirschsprung disease (HSCR) in the Chinese people. We analyzed WNT3A gene (rs61743220, rs192966556 and rs145882986) variants in the whole blood samples from HSCR patients and normal children (control groups). WNT3A expression was also examined by quantitative real-time PCR (qRT-PCR), western blotting and immunostaining. Consequently, when rs192966556 and rs145882986 alleles of the WNT3A gene lack the SNPs, they are especially associated with a greater risk of HSCR (OR [95% confidence interval] = 1.791, p = 0.001; OR [95% confidence interval] = 1.556, p = 0.003, respectively). The mRNA and protein expressions of WNT3A were higher in the aganglionic colon segment tissues than in the normal ganglionic segments tissues. Immunostaining indicates that the staining of WNT3A was much stronger (brown) in the aganglionic colon segment tissues than that in the normal ganglionic colon segment tissues (colorless or light yellow) in the mucous layer and muscular layer. Although preliminary, these results suggest that WNT3A may play an important role in the pathogenesis of HSCR.
PMCID: PMC4014216  PMID: 24817932
Hirschsprung disease; WNT3A; polymorphism; gene and protein; expression
18.  BRAFV600E Mutation and Its Association with Clinicopathological Features of Colorectal Cancer: A Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(3):e90607.
Background
Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC.
Methods
We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs) with 95% confidence intervals (CIs) for each study using a fixed effects or random effects model.
Results
25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955). The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI), CpG island methylator phenotype (CIMP). This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1) methylation.
Conclusions
This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.
doi:10.1371/journal.pone.0090607
PMCID: PMC3940924  PMID: 24594804
19.  High-Resolution Melting Analysis for accurate detection of BRAF mutations: a systematic review and meta-analysis 
Scientific Reports  2014;4:4168.
The high-resolution melting curve analysis (HRMA) might be a good alternative method for rapid detection of BRAF mutations. However, the accuracy of HRMA in detection of BRAF mutations has not been systematically evaluated. We performed a systematic review and meta-analysis involving 1324 samples from 14 separate studies. The overall sensitivity of HRMA was 0.99 (95% confidence interval (CI) = 0.75–0.82), and the overall specificity was very high at 0.99 (95% CI = 0.94–0.98). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 68.01 (95% CI = 25.33–182.64), 0.06 (95% CI = 0.03–0.11), and1263.76 (95% CI = 393.91–4064.39), respectively. The summary receiver operating characteristic curve for the same data shows an area of 1.00 and a Q* value of 0.97. The high sensitivity and specificity, simplicity, low cost, less labor or time and rapid turnaround make HRMA a good alternative method for rapid detection of BRAF mutations in the clinical practice.
doi:10.1038/srep04168
PMCID: PMC3933866  PMID: 24566771
20.  Indices of von Willebrand Factor as Biomarkers of Aortic Stenosis Severity: (From the Biomarkers of Aortic Stenosis Severity [BASS] Study) 
The American journal of cardiology  2012;111(3):374-381.
We correlated von Willebrand factor activity indices and brain natriuretic peptide (BNP) with measures of aortic stenosis (AS) severity, bleeding, symptoms, and freedom from death or aortic valve replacement. Patients with AS (n=66 [16 mild, 20 moderate, 30 severe]) and aortic valve replacement (n=21) were assessed with VWF antigen (VWF:Ag), VWF latex agglutination immunoturbidic activity (VWF:Ltx), platelet function analyzer collagen plus adenosine diphosphate (PFA-CADP), VWF multimer ratio (VWF:mult ratio), and BNP level after echocardiography. In AS patients, mean gradient correlated with BNP (Spearman r=0.29, P=.02), VWF:Ltx/VWF:Ag (r=–0.41, P<.001), PFA-CADP (r=0.49, P<.001), and VWF:mult ratio (r=–0.76, P<.001). The area under the curve (95% CI) for detection of severe AS was 0.62 (0.48-0.77) by elevated BNP, 0.81 (0.69-0.92) by PFA-CADP closure time, 0.69 (0.55-0.82) by VWF:Ltx/VWF:Ag ratio, and 0.86 (0.76-0.95) by VWF:mult ratio. For VWF:mult ratio, a threshold of 0.15 yielded sensitivity and specificity for severe AS of 77% and positive predictive value of 74%. Bleeding (in 14%) was associated with prolonged PFA-CADP and reduced VWF:Ltx/VWF:Ag. Symptoms were associated with elevated BNP and low Duke Activity Status Index score. In 66 AS patients, freedom from death (n=4) or aortic valve replacement (n=22) was associated with PFA-CADP (P=.003), VWF high molecular weight multimers (HMWM) (P=.009), and VWF:Ltx/VWF:Ag (P<.001), but not BNP (P=.32). In severe AS vs aortic valve replacement, PFA-CADP and VWF:mult ratio differed (P<.001), but BNP and VWF:Ltx/VWF:Ag did not. In conclusion, VWF activity indices are associated with AS severity and bleeding, and predictive of cardiovascular outcome.
doi:10.1016/j.amjcard.2012.10.015
PMCID: PMC3932487  PMID: 23168287
aortic stenosis; von Willebrand factor; brain natriuretic peptide; aortic valve replacement
21.  Prognostic significance of neutrophil-lymphocyte ratio in hepatocellular carcinoma: a meta-analysis 
BMC Cancer  2014;14:117.
Backgrounds
Neutrophil-lymphocyte ratio (NLR) has recently been reported as a predictor of Hepatocellular carcinoma (HCC). However, its prognostic value in HCC still remains controversial. In this study, we aimed to evaluate the association between NLR and clinical outcome of HCC patients by performing meta-analysis.
Methods
A comprehensive literature search for relevant studies published up to August 2013 was performed by using PubMed, Ovid, the Cochrane Library and Web of Science databases. Meta-analysis was performed using hazard ratio (HR) or odds ratio (OR) and 95% confidence intervals (95% CIs) as effect measures.
Results
A total of 15 studies encompassing 3094 patients were included in this meta-analysis. Our pooled results showed that high NLR was associated with poor overall survival (OS) and disease free survival (DFS) in HCC initially treated by liver transplantation (HR = 3.42, 95% CI:2.41-4.85,P = 0.000; HR = 5.90, 95% CI:3.99-8.70,P = 0.000, respectively) and surgical resection (HR = 3.33, 95% CI:2.23-4.98, P = 0.000; HR = 2.10, 95% CI: 2.06–2.14, respectively). High NLR was also associated with poor OS in HCC treated by radiofrequency-ablation (HR = 1.28, 95%CI: 1.10-1.48, P = 0.000), TACE (HR = 2.52, 95% CI: 1.64-3.86, P = 0.000) and mixed treatment (HR = 1.85, 95%  CI: 1.40-2.44, P = 0.000), respectively. In addition, high NLR was significantly correlated with the presence of vascular invasion (OR = 2.69, 95% CI: 2.01–3.59, P = 0.000), tumor multifocality (OR = 1.74, 95% CI: 1.30–2.34, P = 0.000) and higher incidence of AFP ≥ 400 ng/ml (OR = 1.46, 95% CI: 1.01–2.09, P = 0.04).
Conclusion
Elevated NLR indicates a poor prognosis for patients with HCC. NLR may be a convenient, easily-obtained, low cost and reliable biomarker with prognostic potential for HCC.
doi:10.1186/1471-2407-14-117
PMCID: PMC4015698  PMID: 24559042
Neutrophil-lymphocyte ratio; Hepatocellular carcinoma; Prognosis
22.  Chinese Herbal Medicine for Aspirin Resistance: A Systematic Review of Randomized Controlled Trials 
Aspirin resistance (AR) is a prevalent phenomenon and leads to significant clinical consequences, but the current evidence for effective interventional strategy is insufficient. The objective of this systematic review is thus to assess the efficacy and safety of Chinese herbal medicine (CHM) for AR. A systematical literature search was conducted in 6 databases until December 2012 to identify randomized controlled trials (RCTs) of CHM for AR. As a result, sixteen RCTs with a total of 1011 subjects were identified, suggesting that the interests of the medical profession and the public in the use of CHM for AR have grown considerably in the recent years. Tongxinluo capsule and Danshen-based prescriptions were the most frequently used herbal prescriptions, while danshen root, milkvetch root, Leech, and Rosewood were the most frequently used single herbs. Despite the apparent reported positive findings, it is premature to determine the efficacy and safety of CHM for the treatment of AR due to poor methodological quality and insufficient safety data. However, CHMs appeared to be well tolerated in all included studies. Thus, CHM as a promising candidate is worthy of improvement and development for further clinical AR trials. Large sample-size and well-designed rigorous RCTs are needed.
doi:10.1155/2014/890950
PMCID: PMC3950618  PMID: 24701247
23.  Discontinuation of antithrombotic therapy for a year or more in patients with continuous-flow left ventricular assist devices☆ 
The recommended anticoagulation regimen during continuous-flow axial left ventricular assist device (LVAD) support is aspirin and warfarin with a targeted international normalized ratio of 2.0–3.0. We report two patients in whom recurrent gastrointestinal bleeding during LVAD support necessitated discontinuation of this anti-thrombotic regimen for a year or more. Despite this, neither patients developed thrombotic complications during 29 patient-months of follow-up. An acquired von Willebrand factor (VWF) abnormality reflected by the absence or decreased abundance of the highest molecular weight multimers was demonstrated in both patients. The gold standard test for platelet function, light transmission platelet aggregometry was measured in one patient and was normal, indicative that the predominant abnormality in the coagulation profile of these patients is an acquired VWF syndrome. Clinical trials are required to address the question whether it is safe to discontinue anticoagulation in LVAD patients with acquired VWF abnormalities.
doi:10.1510/icvts.2010.240747
PMCID: PMC3927556  PMID: 20639306
Heart failure; Ventricular assist device; Anticoagulation; Gastrointestinal bleeding
24.  The impact of heatwaves on mortality in Australia: a multicity study 
BMJ Open  2014;4(2):e003579.
Objectives
To assess the heterogeneity of heatwave-related impacts on mortality across different cities.
Design
A multicity time series study.
Setting
3 largest Australian cities: Brisbane, Melbourne and Sydney.
Participants
All residents living in these cities.
Main outcome measures
Non-external causes mortality data by gender and two age groups (ie, 0–75 and 75+) for these cities during the period 1988–2009 were obtained from relevant government agencies.
Results
Total mortality increased mostly within the same day (lag 0) or a lag of 1 day (lag 1) during almost all heatwaves in three cities. Using the heatwave definition (HWD) as the 95th centile of mean temperature for two or more consecutive days in the summer season, the relative risk for total mortality at lag 1 in Brisbane, Melbourne and Sydney was 1.13 (95% CI 1.08 to 1.19), 1.10 (95% CI 1.06 to 1.14) and 1.06 (95% CI 1.01 to 1.10), respectively. Using the more stringent HWD—the 99th centile of mean temperature for two or more consecutive days, the relative risk of total mortality at the lags of 0–2 days in Brisbane and Melbourne was 1.40 (95% CI 1.29 to 1.51) and 1.47 (95% CI 1.36 to 1.59), respectively. Elderly, particularly females, were more vulnerable to the impact of heatwaves.
Conclusions
A consistent and significant increase in mortality was observed during heatwaves in the three largest Australian cities, but the impacts of heatwave appeared to vary with age, gender, the HWD and geographical area.
doi:10.1136/bmjopen-2013-003579
PMCID: PMC3931989  PMID: 24549159
Climate Changes; Heatwaves; Mean Temperature; Mortality; Time Series Analysis
25.  Prognostic significance of glypican-3 in hepatocellular carcinoma: a meta-analysis 
BMC Cancer  2014;14:104.
Backgrounds
Glypican-3(GPC3) has been implicated in tumor development and progression for several years. However, the prognostic significance of GPC3 expression in patients with hepatocellular carcinoma (HCC) is controversial. We performed a meta-analysis of available studies to assess whether GPC3 can be used as a prognostic factor in patients with HCC.
Methods
We searched PubMed and Ovid EBM Reviews databases and evaluated the reference list of relevant articles for studies that assessed the prognostic relevance of GPC3 in patients with HCC. Meta-analysis was performed using hazard ratio (HR) or odds ratio (OR) and 95% confidence intervals (95% CIs) as effect measures.
Results
A meta-analysis of eight studies included 1070 patients was carried out to evaluate the association between GPC3 and overall survival (OS) and disease-free survival (DFS) in HCC patients. The relation between GPC3 and tumor pathological features was also assessed. Our analysis results indicated that high GPC3 expression predicted poor OS (HR: 1.96, 95% CI: 1.51–2.55) and DFS (HR: 1.99, 95% CI: 1.57-2.51) of patients with HCC. GPC3 overexpression was significantly associated with high tumor grade (OR: 3.30, 95% CI: 2.04–5.33), late TNM stage (OR: 2.26, 95% CI: 1.00–5.12), and the presence of vascular invasion (OR: 2.43, 95% CI: 1.23–4.82).
Conclusions
GPC3 overexpression indicates a poor prognosis for patients with HCC, and it may also have predictive potential for HCC invasion and metastasis.
doi:10.1186/1471-2407-14-104
PMCID: PMC3984430  PMID: 24548704
Glypican-3; Hepatocellular carcinoma; Prognosis

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