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1.  Critical appraisal of the role of gefitinib in the management of locally advanced or metastatic non-small cell lung cancer 
OncoTargets and therapy  2014;7:841-852.
Past studies have demonstrated that epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors can significantly improve clinical outcomes in patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) and sensitive EGFR gene mutations. Gefitinib (Iressa®), the first oral EGFR tyrosine kinase inhibitor, has been shown to be more effective and better tolerated than chemotherapy either in first-line or second-line treatment for patients with advanced NSCLC harboring sensitive EGFR mutations. Conversely, among patients with wild-type EGFR, gefitinib is inferior to standard chemotherapy in both the first-line and second-line settings. Further, gefitinib is effective in patients with brain metastases because of its low molecular weight and excellent penetration of the blood–brain barrier. In this review, we summarize the current data from clinical trials with gefitinib and appraise its role in the management of locally advanced or metastatic NSCLC.
doi:10.2147/OTT.S34124
PMCID: PMC4045263  PMID: 24920926
gefitinib; non-small cell lung cancer; epidermal growth factor receptor; tyrosine kinase inhibitor
2.  Detection of activated KRAS from cancer patient peripheral blood using a weighted enzymatic chip array 
Background
The KRAS oncogene was one of the earliest discoveries of genetic alterations in colorectal and lung cancers. Moreover, KRAS somatic mutations might be used for predicting the efficiency of anti-EGFR therapeutic drugs. The purpose of this research was to improve Activating KRAS Detection Chip by using a weighted enzymatic chip array (WEnCA) platform to detect activated KRAS mutations status in the peripheral blood of non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC) patients in Taiwan.
Methods
Our laboratory developed an Activating KRAS Detection Chip and a WEnCA technique that can detect activated KRAS mutation status by screening circulating cancer cells in the surrounding bloodstream. We collected 390 peripheral blood samples of NSCLC patients (n = 210) and CRC patients (n = 180) to evaluate clinical KRAS activation using this gene array diagnosis apparatus, an Activating KRAS Detection Chip and a WEnCA technique. Subsequently, we prospectively enrolled 88 stage III CRC patients who received adjuvant FOLFOX-4 chemotherapy with or without cetuximab. We compared the chip results of preoperative blood specimens and their relationship with disease control status in these patients.
Results
After statistical analysis, the sensitivity of WEnCA was found to be 93%, and the specificity was found to be 94%. Relapse status and chip results among the stage III CRC patients receiving FOLFOX-4 plus cetuximab (n = 59) and those receiving FOLFOX-4 alone (n = 29) were compared. Among the 51 stage III CRC patients with chip negative results who were treated with FOLFOX-4 plus cetuximab chemotherapy, the relapse rate was 33.3%; otherwise, the relapse rate was 48.5% among the 23 out of 88 patients with chip negative results who received FOLFOX-4 alone. Negative chip results were significantly associated to better treatment outcomes in the FOLFOX-4 plus cetuximab group (P = 0.047).
Conclusions
The results demonstrated that the WEnCA technique is a sensitive and convenient technique that produces easy-to-interpret results for detecting activated KRAS from the peripheral blood of cancer patients. We suggest that the WEnCA technique is also a potential tool for predicting responses in CRC patients following FOLFOX-4 plus cetuximab chemotherapy.
doi:10.1186/1479-5876-12-147
PMCID: PMC4055935  PMID: 24884535
Colorectal cancer; Lung cancer; Peripheral blood; Weighted enzymatic chip array (WEnCA); Activating KRAS Detection Chip
3.  T Cell Factor 1 Is Required for Group 2 Innate Lymphoid Cell Generation 
Immunity  2013;38(4):694-704.
SUMMARY
Group 2 innate lymphoid cells (ILC2) are innate lymphocytes that confer protective type 2 immunity during helminth infection and are also involved in allergic airway inflammation. Here we report that ILC2 development required T cell factor 1 (TCF-1, the product of the Tcf7 gene), a transcription factor also implicated in T cell lineage specification. Tcf7−/− mice lack ILC2, and were unable to mount ILC2-mediated innate type 2 immune responses. Forced expression of TCF-1 in bone marrow progenitors partially bypassed the requirement for Notch signaling in the generation of ILC2 in vivo. TCF-1 acted through both GATA-3-dependent and GATA-3-independent pathways to promote the generation of ILC2. These results are reminiscent of the critical roles of TCF-1 in early T cell development. Hence, transcription factors that underlie early steps of T cell development are also implicated in the development of innate lymphoid cells.
doi:10.1016/j.immuni.2012.12.003
PMCID: PMC4029843  PMID: 23601684
4.  Growth attenuation under saline stress is mediated by the heterotrimeric G protein complex 
BMC Plant Biology  2014;14:129.
Background
Plant growth is plastic, able to rapidly adjust to fluctuation in environmental conditions such as drought and salinity. Due to long-term irrigation use in agricultural systems, soil salinity is increasing; consequently crop yield is adversely affected. It is known that salt tolerance is a quantitative trait supported by genes affecting ion homeostasis, ion transport, ion compartmentalization and ion selectivity. Less is known about pathways connecting NaCl and cell proliferation and cell death. Plant growth and cell proliferation is, in part, controlled by the concerted activity of the heterotrimeric G-protein complex with glucose. Prompted by the abundance of stress-related, functional annotations of genes encoding proteins that interact with core components of the Arabidopsis heterotrimeric G protein complex (AtRGS1, AtGPA1, AGB1, and AGG), we tested the hypothesis that G proteins modulate plant growth under salt stress.
Results
Na+ activates G signaling as quantitated by internalization of Arabidopsis Regulator of G Signaling protein 1 (AtRGS1). Despite being components of a singular signaling complex loss of the Gβ subunit (agb1-2 mutant) conferred accelerated senescence and aborted development in the presence of Na+, whereas loss of AtRGS1 (rgs1-2 mutant) conferred Na+ tolerance evident as less attenuated shoot growth and senescence. Site-directed changes in the Gα and Gβγ protein-protein interface were made to disrupt the interaction between the Gα and Gβγ subunits in order to elevate free activated Gα subunit and free Gβγ dimer at the plasma membrane. These mutations conferred sodium tolerance. Glucose in the growth media improved the survival under salt stress in Col but not in agb1-2 or rgs1-2 mutants.
Conclusions
These results demonstrate a direct role for G-protein signaling in the plant growth response to salt stress. The contrasting phenotypes of agb1-2 and rgs1-2 mutants suggest that G-proteins balance growth and death under salt stress. The phenotypes of the loss-of-function mutations prompted the model that during salt stress, G activation promotes growth and attenuates senescence probably by releasing ER stress.
doi:10.1186/1471-2229-14-129
PMCID: PMC4061919  PMID: 24884438
5.  Synthesis and structure of undoped and indium-doped thermoelectric lead telluride nanoparticles 
Nanoscale Research Letters  2014;9(1):227.
Undoped and indium (In)-doped lead telluride (PbTe) nanostructures were synthesized via solvothermal/hydrothermal route. The crystalline structure of the as-prepared undoped and In-doped PbTe samples was examined by X-ray diffraction (XRD) which indicated the formation of face-centered single-phase cubic crystal. A first principle calculation on indium doping shows that the indium atoms are more likely to replace lead (Pb) rather than to take the interstitial sites. Laser-induced breakdown spectroscopy (LIBS) analysis confirms that indium is incorporated into the PbTe matrix of the indium-doped PbTe samples. The effects of surfactant and synthesis temperature on the structure and morphology of the undoped PbTe were also investigated; it was found that PbTe nanostructures synthesized with the addition of surfactants exhibited uniform shapes and their size increased with the synthesis temperature.
doi:10.1186/1556-276X-9-227
PMCID: PMC4022418  PMID: 24872808
Lead telluride; Nanostructure; Solvothermal/hydrothermal synthesis; First principle calculation
6.  Genome-wide identification of heat shock proteins (Hsps) and Hsp interactors in rice: Hsp70s as a case study 
BMC Genomics  2014;15(1):344.
Background
Heat shock proteins (Hsps) perform a fundamental role in protecting plants against abiotic stresses. Although researchers have made great efforts on the functional analysis of individual family members, Hsps have not been fully characterized in rice (Oryza sativa L.) and little is known about their interactors.
Results
In this study, we combined orthology-based approach with expression association data to screen rice Hsps for the expression patterns of which strongly correlated with that of heat responsive probe-sets. Twenty-seven Hsp candidates were identified, including 12 small Hsps, six Hsp70s, three Hsp60s, three Hsp90s, and three clpB/Hsp100s. Then, using a combination of interolog and expression profile-based methods, we inferred 430 interactors of Hsp70s in rice, and validated the interactions by co-localization and function-based methods. Subsequent analysis showed 13 interacting domains and 28 target motifs were over-represented in Hsp70s interactors. Twenty-four GO terms of biological processes and five GO terms of molecular functions were enriched in the positive interactors, whose expression levels were positively associated with Hsp70s. Hsp70s interaction network implied that Hsp70s were involved in macromolecular translocation, carbohydrate metabolism, innate immunity, photosystem II repair and regulation of kinase activities.
Conclusions
Twenty-seven Hsps in rice were identified and 430 interactors of Hsp70s were inferred and validated, then the interacting network of Hsp70s was induced and the function of Hsp70s was analyzed. Furthermore, two databases named Rice Heat Shock Proteins (RiceHsps) and Rice Gene Expression Profile (RGEP), and one online tool named Protein-Protein Interaction Predictor (PPIP), were constructed and could be accessed at http://bioinformatics.fafu.edu.cn/.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-344) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-344
PMCID: PMC4035072  PMID: 24884676
Rice (Oryza sativa L.); Heat shock proteins; Genome wide; Identification
7.  SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1 
Molecular Cancer  2014;13:95.
Background
Grb2 (Growth factor receptor-bound protein 2) is a key adaptor protein in maintaining the ERK activity via linking Sos1 (Son of sevenless homolog 1) or other proteins to activated RTKs, such as EGFR. Currently, little knowledge is available concerning the post-translational modification (PTM) of Grb2 except for its phosphorylation. Since emerging evidences have highlighted the importance of SUMOylation (Small ubiquitin-related modifier), a reversible PTM, in modulating protein functions, we wondered if Grb2 could be SUMOylated and thereby influences its functions especially involved in the Ras/MEK/ERK pathway.
Methods
SUMOylation of Grb2 was analyzed with the in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown and the in vitro E.coli-based SUMOylation assay. To test the ERK activity and cell transformation, the murine fibroblast cell line NIH/3T3 and the murine colon cancer cell line CMT-93 were used for the experiments including Grb2 knockdown, ectopic re-expression, cell transformation and migration. Immunoprecipitation (IP) was employed for seeking proteins that interact with SUMO modified Grb2. Xenograft tumor model in mice was conducted to verify that Grb2 SUMOylation regulated tumorigenesis in vivo.
Results
Grb2 can be SUMOylated by SUMO1 at lysine 56 (K56), which is located in the linker region between the N-terminal SH3 domain and the SH2 domain. Knockdown of Grb2 reduced the ERK activity and suppressed cell motility and tumorigenesis in vitro and in vivo, which were all rescued by stable ectopic re-expression of wild-type Grb2 but not the mutant Grb2K56R. Furthermore, Grb2 SUMOylation at K56 increased the formation of Grb2-Sos1 complex, which sequentially leads to the activation of Ras/MEK/MAPK pathway.
Conclusions
Our results provide evidences that Grb2 is SUMOylated in vivo and this modification enhances ERK activities via increasing the formation of Grb2-Sos1 complex, and may consequently promote cell motility, transformation and tumorigenesis.
doi:10.1186/1476-4598-13-95
PMCID: PMC4021559  PMID: 24775912
Grb2; SUMOylation; Sos1; ERK activity; Tumorigenesis; Cell migration
8.  Hepatoprotective effect of Cichorium intybus L., a traditional Uighur medicine, against carbon tetrachloride-induced hepatic fibrosis in rats 
AIM: To investigate the hepatoprotective effect of a Cichorium intybus L. extract (CIE) on CCl4-induced hepatic fibrosis in rats.
METHODS: Seventy-two male Wistar albino rats were randomly divided into six groups of twelve rats each. The normal control group was allowed free access to food and water. Liver injury was performed in the remaining five groups with an i.p. injection of a 1.0 mL/kg CCl4 and olive oil (2:3 v/v) mixture, twice weekly for 8 weeks. All rats, with the exception of the injury model group, were intragastrically (i.g.,) administered quantum satis (q.s.) dosages [CIE group: 6, 18, and 54 mg/kg, respectively; Fu Fang Bie Jia Ruan Gan Pian (FFBJRGP) group: 780 mg/kg]. The oral administration of different drugs was performed on the day before CCl4 administration and subsequently once per day for 8 wk. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hexadecenoic acid (HA), laminin (LN), hydroxyproline (Hyp), and glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD) in the rat livers were measured. Histopathological changes in the liver were assessed for each group using HE staining and a Masson Trichrome examination. The expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) was examined by immunohistochemical analysis.
RESULTS: CIE at oral doses of 6, 18, and 54 g/kg per day showed a significant hepatoprotective effect, especially at a dose of 54 g/kg per day. CIE doses reduced the levels of AST (149.04 ± 34.44, P < 0.01), ALT (100.72 ± 27.19, P < 0.01), HA (548.50 ± 65.09, P < 0.01), LN (28.69 ± 3.32, P < 0.01) and Hyp (263.33 ± 75.82, P < 0.01). With regards to hepatoprotective activity, the CIE dose of 54 g/kg per day produced the largest significant effect by increasing GSH (3.11 ± 0.81), SOD (269.98 ± 33.77, P < 0.01) and reducing MDA (2.76 ± 0.51, P < 0.01) levels in the liver. The expressions of TGF-β1 and α-SMA were measured by immunohistology and found to be significantly reduced by CIE in a dose-dependent manner.
CONCLUSION: CIE may effectively protect against CCl4-induced hepatic fibrosis in rats; thus, it is a promising anti-fibrotic therapeutic agent.
doi:10.3748/wjg.v20.i16.4753
PMCID: PMC4000513  PMID: 24782629
Cichorium intybus L. extract; Traditional Uighur medicine; Hepatic fibrosis; Carbon tetrachloride
9.  The Histone Mark H3K36me3 Regulates Human DNA Mismatch Repair through its Interaction with MutSα 
Cell  2013;153(3):590-600.
Summary
DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 tri-methyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.
doi:10.1016/j.cell.2013.03.025
PMCID: PMC3641580  PMID: 23622243
10.  Different Relationships between Temporal Phylogenetic Turnover and Phylogenetic Similarity and in Two Forests Were Detected by a New Null Model 
PLoS ONE  2014;9(4):e95703.
Background
Ecologists have been monitoring community dynamics with the purpose of understanding the rates and causes of community change. However, there is a lack of monitoring of community dynamics from the perspective of phylogeny.
Methods/Principle Findings
We attempted to understand temporal phylogenetic turnover in a 50 ha tropical forest (Barro Colorado Island, BCI) and a 20 ha subtropical forest (Dinghushan in southern China, DHS). To obtain temporal phylogenetic turnover under random conditions, two null models were used. The first shuffled names of species that are widely used in community phylogenetic analyses. The second simulated demographic processes with careful consideration on the variation in dispersal ability among species and the variations in mortality both among species and among size classes. With the two models, we tested the relationships between temporal phylogenetic turnover and phylogenetic similarity at different spatial scales in the two forests. Results were more consistent with previous findings using the second null model suggesting that the second null model is more appropriate for our purposes. With the second null model, a significantly positive relationship was detected between phylogenetic turnover and phylogenetic similarity in BCI at a 10 m×10 m scale, potentially indicating phylogenetic density dependence. This relationship in DHS was significantly negative at three of five spatial scales. This could indicate abiotic filtering processes for community assembly. Using variation partitioning, we found phylogenetic similarity contributed to variation in temporal phylogenetic turnover in the DHS plot but not in BCI plot.
Conclusions/Significance
The mechanisms for community assembly in BCI and DHS vary from phylogenetic perspective. Only the second null model detected this difference indicating the importance of choosing a proper null model.
doi:10.1371/journal.pone.0095703
PMCID: PMC3991709  PMID: 24748022
11.  Systematic Identification of Cell-Wall Related Genes in Populus Based on Analysis of Functional Modules in Co-Expression Network 
PLoS ONE  2014;9(4):e95176.
The identification of novel genes relevant to plant cell wall (PCW) biosynthesis in Populus is a highly important and challenging problem. We surveyed candidate Populus cell wall genes using a non-targeted approach. First, a genome-wide Populus gene co-expression network (PGCN) was constructed using microarray data available in the public domain. Module detection was then performed, followed by gene ontology (GO) enrichment analysis, to assign the functional category to these modules. Based on GO annotation, the modules involved in PCW biosynthesis were then selected and analyzed in detail to annotate the candidate PCW genes in these modules, including gene annotation, expression of genes in different tissues, and so on. We examined the overrepresented cis-regulatory elements (CREs) in the gene promoters to understand the possible transcriptionally co-regulated relationships among the genes within the functional modules of cell wall biosynthesis. PGCN contains 6,854 nodes (genes) with 324,238 edges. The topological properties of the network indicate scale-free and modular behavior. A total of 435 modules were identified; among which, 67 modules were identified by overrepresented GO terms. Six modules involved in cell wall biosynthesis were identified. Module 9 was mainly involved in cellular polysaccharide metabolic process in the primary cell wall, whereas Module 4 comprises genes involved in secondary cell wall biogenesis. In addition, we predicted and analyzed 10 putative CREs in the promoters of the genes in Module 4 and Module 9. The non-targeted approach of gene network analysis and the data presented here can help further identify and characterize cell wall related genes in Populus.
doi:10.1371/journal.pone.0095176
PMCID: PMC3988181  PMID: 24736620
12.  Identification of Microbial Communities in Open and Closed Circuit Bioelectrochemical MBRs by High-Throughput 454 Pyrosequencing 
PLoS ONE  2014;9(4):e93842.
Two bioelectrochemical membrane bioreactors (MBRs) developed by integrating microbial fuel cell and MBR technology were operated under closed-circuit and open-circuit modes, and high-throughput 454 pyrosequencing was used to investigate the effects of the power generation on the microbial community of bio-anode and bio-cathode. Microbes on the anode under open-circuit operation (AO) were enriched and highly diverse when compared to those on the anode under closed-circuit operation (AC). However, among the cathodes the closed-circuit mode (CC) had richer and more diverse microbial community compared to the cathode under open-circuit mode (CO). On the anodes AO and AC, Proteobacteria and Bacteroidetes were the dominant phyla, while Firmicutes was enriched only on AC. Deltaproteobacteria affiliated to Proteobacteria were also more abundant on AC than AO. Furthermore, the relative abundance of Desulfuromonas, which are well-known electrogenic bacteria, were much higher on AC (10.2%) when compared to AO (0.11%), indicating that closed-circuit operation was more conducive for the growth of electrogenic bacteria on the anodes. On the cathodes, Protebacteria was robust on CC while Bacteroidetes was more abundant on CO. Rhodobacter and Hydrogenophaga were also enriched on CC than CO, suggesting that these genera play a role in electron transfer from the cathode surface to the terminal electron acceptors in the bioelectrochemical MBR under closed-circuit operation.
doi:10.1371/journal.pone.0093842
PMCID: PMC3976363  PMID: 24705450
13.  Mutation Spectrum of Six Genes in Chinese Phenylketonuria Patients Obtained through Next-Generation Sequencing 
PLoS ONE  2014;9(4):e94100.
Background
The identification of gene variants plays an important role in the diagnosis of genetic diseases.
Methodology/Principal Findings
To develop a rapid method for the diagnosis of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficiency, we designed a multiplex, PCR-based primer panel to amplify all the exons and flanking regions (50 bp average) of six PKU-associated genes (PAH, PTS, GCH1, QDPR, PCBD1 and GFRP). The Ion Torrent Personal Genome Machine (PGM) System was used to detect mutations in all the exons of these six genes. We tested 93 DNA samples from blood specimens from 35 patients and their parents (32 families) and 26 healthy adults. Using strict bioinformatic criteria, this sequencing data provided, on average, 99.14% coverage of the 39 exons at more than 70-fold mean depth of coverage. We found 23 previously documented variants in the PAH gene and six novel mutations in the PAH and PTS genes. A detailed analysis of the mutation spectrum of these patients is described in this study.
Conclusions/Significance
These results were confirmed by Sanger sequencing. In conclusion, benchtop next-generation sequencing technology can be used to detect mutations in monogenic diseases and can detect both point mutations and indels with high sensitivity, fidelity and throughput at a lower cost than conventional methods in clinical applications.
doi:10.1371/journal.pone.0094100
PMCID: PMC3976377  PMID: 24705691
14.  Umbilical metastasis derived from early stage rectal cancer: a case report 
Background
Umbilical metastasis, also called Sister Mary Joseph’s nodule (SMJN), is defined as the umbilical nodule associated with advanced metastatic intra-abdominal and pelvic malignancies. A patient with umbilical metastasis has been deemed to have a poor prognosis. Rectal cancer presenting with a SMJN is a rare phenomenon, especially in the early stage and in middle-low rectal cancer.
Case presentation
We report a case of a 70-year-old male presenting with umbilical metastasis derived from rectal cancer (10 cm from the anal verge, T2N0).
Discussion and conclusion
For rectal cancer with umbilical metastasis, the exact metastatic routes as well as the criterion of diagnosis and treatments are not very clear. Here we review the literature on rectal cancer and SMJN to deepen the understanding of this disease.
doi:10.1186/1477-7819-12-82
PMCID: PMC3992153  PMID: 24708697
Rectal cancer; Umbilical metastasis; Sister Mary Joseph’s nodule (SMJN)
15.  Incorporating group correlations in genome-wide association studies using smoothed group Lasso 
Biostatistics (Oxford, England)  2012;14(2):205-219.
In genome-wide association studies, penalization is an important approach for identifying genetic markers associated with disease. Motivated by the fact that there exists natural grouping structure in single nucleotide polymorphisms and, more importantly, such groups are correlated, we propose a new penalization method for group variable selection which can properly accommodate the correlation between adjacent groups. This method is based on a combination of the group Lasso penalty and a quadratic penalty on the difference of regression coefficients of adjacent groups. The new method is referred to as smoothed group Lasso (SGL). It encourages group sparsity and smoothes regression coefficients for adjacent groups. Canonical correlations are applied to the weights between groups in the quadratic difference penalty. We first derive a GCD algorithm for computing the solution path with linear regression model. The SGL method is further extended to logistic regression for binary response. With the assistance of the majorize–minimization algorithm, the SGL penalized logistic regression turns out to be an iteratively penalized least-square problem. We also suggest conducting principal component analysis to reduce the dimensionality within groups. Simulation studies are used to evaluate the finite sample performance. Comparison with group Lasso shows that SGL is more effective in selecting true positives. Two datasets are analyzed using the SGL method.
doi:10.1093/biostatistics/kxs034
PMCID: PMC3590928  PMID: 22988281
Group selection; Regularization; SNP; Smoothing
16.  An Ascending Ramp Biphasic Waveform Has a Lower Defibrillation Threshold and Releases Less Troponin I Than a Truncated Exponential Biphasic Waveform 
Circulation  2012;126(11):1328-1333.
Background
We tested the hypothesis that the shape of the shock waveform affects not only the defibrillation threshold (DFT), but also the amount of cardiac damage.
Methods and Results
DFTs were determined for 11 waveforms: 3 ascending ramp, 3 descending ramp, and 3 rectilinear first phase biphasic waveforms, a Gurvich waveform, and a truncated exponential biphasic waveform in 6 pigs with electrodes in the RV apex and SVC. The ascending, descending and rectilinear waveforms had 4, 8 and 16 ms 1st phases and a 3.5 ms 2nd rectilinear phase half the voltage of the 1st phase. The exponential biphasic waveform had a 60% 1st phase and a 50% 2nd phase tilt. In a second study, we attempted to defibrillate after 10 s of VF with a single ≈ 30 J shock (6 pigs successfully defibrillated with 8 ms ascending, 8 ms rectilinear wave and truncated exponential biphasic waveforms). Troponin I blood levels were determined before and 2 to 10 hrs after the shock. The lowest energy DFT was for the 8 ms ascending ramp (14.6±7.3 SD J), which was significantly less than for the truncated exponential (19.6±6.3 J). Six hours postshock, troponin I in ng/ml was significantly less for the ascending ramp (0.80±0.54) than for the truncated exponential (1.92±0.47) or the rectilinear waveform (1.17 ±0.45).
Conclusions
The ascending ramp has a significantly lower DFT, and at ≈ 30 J causes 58% less troponin I release than the truncated exponential biphasic shock. Therefore, the shock waveform affects both the DFT and the amount of cardiac damage.
doi:10.1161/CIRCULATIONAHA.112.109777
PMCID: PMC3968773  PMID: 22865891
cardioversion; defibrillation; troponin; ventricular fibrillation
17.  Effects of Huanglian-Jie-Du-Tang and Its Modified Formula on the Modulation of Amyloid-β Precursor Protein Processing in Alzheimer's Disease Models 
PLoS ONE  2014;9(3):e92954.
Huanglian-Jie-Du-Tang (HLJDT) is a famous traditional Chinese herbal formula that has been widely used clinically to treat cerebral ischemia. Recently, we found that berberine, a major alkaloid compound in HLJDT, reduced amyloid-β (Aβ) accumulation in an Alzheimer’s disease (AD) mouse model. In this study, we compared the effects of HLJDT, four single component herbs of HLJDT (Rhizoma coptidis (RC), Radix scutellariae (RS), Cortex phellodendri (CP) and Fructus gardenia (FG)) and the modified formula of HLJDT (HLJDT-M, which is free of RS) on the regulatory processing of amyloid-β precursor protein (APP) in an in vitro model of AD. Here we show that treatment with HLJDT-M and its components RC, CP, and the main compound berberine on N2a mouse neuroblastoma cells stably expressing human APP with the Swedish mutation (N2a-SwedAPP) significantly decreased the levels of full-length APP, phosphorylated APP at threonine 668, C-terminal fragments of APP, soluble APP (sAPP)-α and sAPPβ-Swedish and reduced the generation of Aβ peptide in the cell lysates of N2a-SwedAPP. HLJDT-M showed more significant APP- and Aβ- reducing effects than berberine, RC or CP treatment alone. In contrast, HLJDT, its component RS and the main active compound of RS, baicalein, strongly increased the levels of all the metabolic products of APP in the cell lysates. The extract from FG, however, did not influence APP modulation. Interestingly, regular treatment of TgCRND8 APP transgenic mice with baicalein exacerbated the amyloid plaque burden, APP metabolism and Aβ production. Taken together, these data provide convincing evidence that HLJDT and baicalein treatment can increase the amyloidogenic metabolism of APP which is at least partly responsible for the baicalein-mediated Aβ plaque increase in the brains of TgCRND8 mice. On the other hand, HLJDT-M significantly decreased all the APP metabolic products including Aβ. Further study of HLJDT-M for therapeutic use in treating AD is warranted.
doi:10.1371/journal.pone.0092954
PMCID: PMC3966845  PMID: 24671102
19.  Increased Complement C1q Level Marks Active Disease in Human Tuberculosis 
PLoS ONE  2014;9(3):e92340.
Background
Complement functions as an important host defense system and complement C5 and C7 have been implicated in immunopathology of tuberculosis. However, little is known about the role of other complement components in tuberculosis.
Methods
Complement gene expression in peripheral blood mononuclear cells of tuberculosis patients and controls were determined using whole genome transcriptional microarray assays. The mRNA and protein levels of three C1q components, C1qA, C1qB, and C1qC, were further validated by qRT-PCR and enzyme-linked immunosorbent assay, respectively. The percentages of C1q expression in CD14 positive cells were determined by flow cytometry. Finally, C1qC protein level was quantified in the pleural fluid of tuberculosis and non-tuberculosis pleurisy.
Results
C1q expression increases significantly in the peripheral blood of patients with active tuberculosis compared to healthy controls and individuals with latent TB infection. The percentage of C1q-expressing CD14 positive cells is significantly increased in active TB patients. C1q expression in the peripheral blood correlates with sputum smear positivity in tuberculosis patients and is reduced after anti-tuberculosis chemotherapy. Notably, receiver operating characteristic analysis showed that C1qC mRNA levels in peripheral blood efficiently discriminate active from latent tuberculosis infection and healthy controls. Additionally, C1qC protein level in pleural effusion shows improved power in discriminating tuberculosis from non-tuberculosis pleurisy when compared to other inflammatory markers, such as IL-6 and TNF-α.
Conclusions
C1q expression correlates with active disease in human tuberculosis. C1q could be a potential diagnostic marker to discriminate active tuberculosis from latent tuberculosis infection as well as tuberculosis pleurisy from non-tuberculosis pleurisy.
doi:10.1371/journal.pone.0092340
PMCID: PMC3960215  PMID: 24647646
20.  Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease 
Nature genetics  2012;44(8):890-894.
We performed a meta-analysis of 2 genome-wide association studies of coronary artery disease comprising 1,515 cases with coronary artery disease and 5,019 controls, followed by de novo replication studies in 15,460 cases and 11,472 controls, all of Chinese Han descent. We successfully identified four new loci for coronary artery disease reaching genome-wide significance (P < 5 × 10−8), which mapped in or near TTC32-WDR35, GUCY1A3, C6orf10-BTNL2 and ATP2B1. We also replicated four loci previously identified in European populations (PHACTR1, TCF21, CDKN2A/B and C12orf51). These findings provide new insights into biological pathways for the susceptibility of coronary artery disease in Chinese Han population.
doi:10.1038/ng.2337
PMCID: PMC3927410  PMID: 22751097
21.  Production of shikimic acid from Escherichia coli through chemically inducible chromosomal evolution and cofactor metabolic engineering 
Background
Shikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu®), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production.
Results
The pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroG fbr , aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (ΔaroKΔaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5Ptac promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol.
Conclusion
An SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was constructed by triclosan-induced chromosomal evolution. We present the first demonstration that increasing NADPH availability by overexpressing the pntAB or nadK genes significantly enhances SA production.
doi:10.1186/1475-2859-13-21
PMCID: PMC3923554  PMID: 24512078
Shikimic acid; Escherichia coli; Chemically induced chromosomal evolution; NADPH; Transhydrogenase; NAD kinase
22.  Effects of bone cement on intervertebral disc degeneration 
Percutaneous vertebroplasty (PVP) is popular for the treatment of intractable pain due to vertebral collapse from various lesions, intervertebral disk leakage of cement is a frequent complication. The aim of this study was to determine whether bone cement causes disc degeneration, and to evaluate the degree of intervertebral disc degeneration (IDD) according to the time period following cement injection, and the type and volume of cement injected. Sixteen dogs were randomly divided into two groups that were sacrificed at 12 or 24 weeks following cement injection. Five intervertebral discs in each dog were studied, including one control untreated disc and four discs randomly injected with polymethylmethacrylate (PMMA) or calcium phosphate cement (CPC) in two quantities. Radiographic and magnetic resonance imaging (MRI) studies were performed prior to animal sacrifice. T2-weighted mid-sagittal images of the discs were qualitatively analyzed for evidence of degeneration by calculating the MRI index, and all harvested discs were studied histopathologically. IDD was not evident in control discs. Univariate analysis revealed significant differences in the MRI index and the histological grade of disc degeneration in terms of the time period following cement injection, as well as the type and volume of cement injected. Result indicate that direct contact with PMMA and CPC can lead to IDD. However, IDD induced by PMMA was more severe than that induced by CPC. The extent of IDD was found to correlate with the time period post-cement injection and the volume of cement injected into the disc. PMMA and CPC may lead to intervertebral disc degeneration. Intervertebral disc degeneration induced by PMMA is more serious than that of CPC. The degree of intervertebral disc degeneration is correlative to the time after operation and the doses of bone cement.
doi:10.3892/etm.2014.1531
PMCID: PMC3965156  PMID: 24669259
vertebroplasty; polymethyl methacrylate; calcium phosphate cement; intervertebral disc; degeneration; magnetic resonance imaging
23.  Rare, Non-Synonymous Variant in the Smooth Muscle-Specific Isoform of Myosin Heavy Chain, MYH11, R247C, Alters Force Generation in the Aorta and Phenotype of Smooth Muscle Cells 
Circulation research  2012;110(11):1411-1422.
Rationale
Mutations in MYH11 cause autosomal dominant inheritance of thoracic aortic aneurysms and dissections. At the same time, rare, non-synonymous variants in MYH11 that are predicted to disrupt protein function but do not cause inherited aortic disease are common in the general population and the vascular disease risk associated with these variants is unknown.
Objective
To determine the consequences of the recurrent MYH11 rare variant, R247C, through functional studies in vitro and analysis of a knock-in mouse model with this specific variant, including assessment of aortic contraction, response to vascular injury, and phenotype of primary aortic smooth muscle cells (SMCs).
Methods and Results
The steady state ATPase activity (actin-activated) and the rates of phosphate and ADP release were lower for the R247C mutant myosin than for the wild-type, as was the rate of actin filament sliding in an in vitro motility assay. Myh11R247C/R247C mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11R247C/R247C mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Primary aortic SMCs explanted from the Myh11R247C/R247C mice were de-differentiated compared with wild-type SMCs based on increased proliferation and reduced expression of SMC contractile proteins. The mutant SMCs also displayed altered focal adhesions and decreased Rho activation, associated with decreased nuclear localization of myocardin-related transcription factor-A. Exposure of the Myh11R247C/R247C SMCs to a Rho activator rescued the de-differentiated phenotype of the SMCs.
Conclusions
These results indicate that a rare variant in MYH11, R247C, alters myosin contractile function and SMC phenotype, leading to increased proliferation in vitro and in response to vascular injury.
doi:10.1161/CIRCRESAHA.111.261743
PMCID: PMC3917690  PMID: 22511748
MYH11; smooth muscle myosin heavy chain; thoracic aortic aneurysms and dissections; smooth muscle differentiation; mouse model
24.  Autophagy in Muscle of Glucose-Infusion Hyperglycemia Rats and Streptozotocin-Induced Hyperglycemia Rats via Selective Activation of m-TOR or FoxO3 
PLoS ONE  2014;9(2):e87254.
Autophagy is a conserved process in eukaryotes required for metabolism and is involved in diverse diseases. To investigate autophagy in skeletal muscle under hyperglycemia status, we established two hyperglycemia-rat models that differ in their circulating insulin levels, by glucose infusion and singe high-dose streptozotocin injection. We then detected expression of autophagy related genes with real-time PCR and western blot. We found that under hyperglycemia status induced by glucose-infusion, autophagy was inhibited in rat skeletal muscle, whereas under streptozotocin-induced hyperglycemia status autophagy was enhanced. Meanwhile, hyperglycemic gastrocnemius muscle was more prone to autophagy than soleus muscle. Furthermore, inhibition of autophagy in skeletal muscle in glucose-infusion hyperglycemia rats was mediated by the m-TOR pathway while m-TOR and FoxO3 both contributed to enhancement of autophagy in gastrocnemius muscle in streptozotocin-induced hyperglycemia rats. These data shows that insulin plays a relatively more important role than hyperglycemia in regulating autophagy in hyperglycemia rat muscle through selectively activating the m-TOR or FoxO3 pathway in a fiber-selective manner.
doi:10.1371/journal.pone.0087254
PMCID: PMC3911944  PMID: 24498304
25.  Incorporating Network Structure in Integrative Analysis of Cancer Prognosis Data 
Genetic epidemiology  2012;37(2):173-183.
In high-throughput cancer genomic studies, markers identified from the analysis of single datasets may have unsatisfactory properties because of low sample sizes. Integrative analysis pools and analyzes raw data from multiple studies, and can effectively increase sample size and lead to improved marker identification results. In this study, we consider the integrative analysis of multiple high-throughput cancer prognosis studies. In the existing integrative analysis studies, the interplay among genes, which can be described using the network structure, has not been effectively accounted for. In network analysis, tightly-connected nodes (genes) are more likely to have related biological functions and similar regression coefficients. The goal of this study is to develop an analysis approach that can incorporate the gene network structure in integrative analysis. To this end, we adopt an AFT (accelerated failure time) model to describe survival. A weighted least squares approach, which has low computational cost, is adopted for estimation. For marker selection, we propose a new penalization approach. The proposed penalty is composed of two parts. The first part is a group MCP penalty, and conducts gene selection. The second part is a Laplacian penalty, and smoothes the differences of coefficients for tightly-connected genes. A group coordinate descent approach is developed to compute the proposed estimate. Simulation study shows satisfactory performance of the proposed approach when there exist moderate to strong correlations among genes. We analyze three lung cancer prognosis datasets, and demonstrate that incorporating the network structure can lead to the identification of important genes and improved prediction performance.
doi:10.1002/gepi.21697
PMCID: PMC3909475  PMID: 23161517
Integrative analysis; Cancer prognosis; Gene network; Penalized selection; Laplacian shrinkage

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