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1.  Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium tuberculosis–Induced Apoptosis in Mice 
PLoS ONE  2010;5(2):e9205.
Background
Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive.
Methodology/Principal Findings
We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-α, IL-1β, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration.
Conclusions/Significance
These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.
doi:10.1371/journal.pone.0009205
PMCID: PMC2824811  PMID: 20174568
2.  Clinical Validation of KRAS, BRAF, and EGFR Mutation Detection Using Next-Generation Sequencing 
Objectives
To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.
Methods
We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA).
Results
We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations.
Conclusions
Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts.
doi:10.1309/AJCPMWGWGO34EGOD
PMCID: PMC4332779  PMID: 24838331
Next-generation sequencing; Validation; KRAS; BRAF; EGFR; Read depth; Deamination
3.  3DProIN: Protein-Protein Interaction Networks and Structure Visualization 
3DProIN is a computational tool to visualize protein–protein interaction networks in both two dimensional (2D) and three dimensional (3D) view. It models protein-protein interactions in a graph and explores the biologically relevant features of the tertiary structures of each protein in the network. Properties such as color, shape and name of each node (protein) of the network can be edited in either 2D or 3D views. 3DProIN is implemented using 3D Java and C programming languages. The internet crawl technique is also used to parse dynamically grasped protein interactions from protein data bank (PDB). It is a java applet component that is embedded in the web page and it can be used on different platforms including Linux, Mac and Window using web browsers such as Firefox, Internet Explorer, Chrome and Safari. It also was converted into a mac app and submitted to the App store as a free app. Mac users can also download the app from our website. 3DProIN is available for academic research at http://bicompute.appspot.com
doi:10.7726/ajbcb.2014.1003
PMCID: PMC4316736  PMID: 25664223
Protein-protein interaction; Tertiary structures; Graph visualization; Protein-protein interaction network
4.  Storage and retrieval of (3 + 1)-dimensional weak-light bullets and vortices in a coherent atomic gas 
Scientific Reports  2015;5:8211.
A robust light storage and retrieval (LSR) in high dimensions is highly desirable for light and quantum information processing. However, most schemes on LSR realized up to now encounter problems due to not only dissipation, but also dispersion and diffraction, which make LSR with a very low fidelity. Here we propose a scheme to achieve a robust storage and retrieval of weak nonlinear high-dimensional light pulses in a coherent atomic gas via electromagnetically induced transparency. We show that it is available to produce stable (3 + 1)-dimensional light bullets and vortices, which have very attractive physical property and are suitable to obtain a robust LSR in high dimensions.
doi:10.1038/srep08211
PMCID: PMC4314635  PMID: 25645119
5.  Nonlinear optical microscopy for immunoimaging: a custom optimized system of high-speed, large-area, multicolor imaging 
Background
The nonlinear optical microscopy has become the current state-of-the-art for intravital imaging. Due to its advantages of high resolution, superior tissue penetration, lower photodamage and photobleaching, as well as intrinsic z-sectioning ability, this technology has been widely applied in immunoimaging for a decade. However, in terms of monitoring immune events in native physiological environment, the conventional nonlinear optical microscope system has to be optimized for live animal imaging. Generally speaking, three crucial capabilities are desired, including high-speed, large-area and multicolor imaging. Among numerous high-speed scanning mechanisms used in nonlinear optical imaging, polygon scanning is not only linearly but also dispersion-freely with high stability and tunable rotation speed, which can overcome disadvantages of multifocal scanning, resonant scanner and acousto-optical deflector (AOD). However, low frame rate, lacking large-area or multicolor imaging ability make current polygonbased nonlinear optical microscopes unable to meet the requirements of immune event monitoring.
Methods
We built up a polygon-based nonlinear optical microscope system which was custom optimized for immunoimaging with high-speed, large-are and multicolor imaging abilities.
Results
Firstly, we validated the imaging performance of the system by standard methods. Then, to demonstrate the ability to monitor immune events, migration of immunocytes observed by the system based on typical immunological models such as lymph node, footpad and dorsal skinfold chamber are shown. Finally, we take an outlook for the possible advance of related technologies such as sample stabilization and optical clearing for more stable and deeper intravital immunoimaging.
Conclusions
This study will be helpful for optimizing nonlinear optical microscope to obtain more comprehensive and accurate information of immune events.
doi:10.3978/j.issn.2223-4292.2014.11.07
PMCID: PMC4312289
Nonlinear optical microscopy; immunoimaging; high-speed; large-area; multicolor
6.  A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation 
Molecular Biology of the Cell  2014;25(23):3798-3812.
The role of phosphorylation in ADAM17-dependent shedding is controversial. We show that the A1 adenosine receptor stimulates exocytosis in umbrella cells by a pathway that requires phosphorylation of ADAM17–Ser-811, followed by HB-EGF shedding and EGF receptor transactivation. Preventing ADAM17 phosphorylation blocks these downstream events.
Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved. Using native bladder epithelium, in some cases transduced with adenoviruses encoding small interfering RNA, we observe that stimulation of apically localized A1 adenosine receptors (A1ARs) triggers a Gi-Gβγ-phospholipase C-protein kinase C (PKC) cascade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A1AR activation. Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17S811A mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17S811A in bladder tissues impairs A1AR-induced apical exocytosis. We conclude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphorylation state of Ser-811 in its cytoplasmic domain.
doi:10.1091/mbc.E14-03-0818
PMCID: PMC4230785  PMID: 25232008
7.  Acupuncture May Exert Its Therapeutic Effect through MicroRNA-339/Sirt2/NFκB/FOXO1 Axis 
BioMed Research International  2015;2015:249013.
Recently, we have found that a number of microRNAs (miRNAs) and proteins are involved in the response to acupuncture therapy in hypertensive rats. Our bioinformatics study suggests an association between these miRNAs and proteins, which include miR-339 and sirtuin 2 (Sirt2). In this paper, we aimed to investigate whether Sirt2 was a direct target of miR-339 in neurons. In human SH-SY5Y cells, the luciferase assay implied that Sirt2 was likely a target of miRNA-339. Overexpression of miR-339 downregulated Sirt2 expression, while knockdown of miR-339 upregulated Sirt2 expression in human SH-SY5Y cells and rat PC12 cells. In addition, overexpression of miR-399 increased the acetylation of nuclear factor-κB (NF-κB) and forkhead box protein O1 (FOXO1) in SH-SY5Y cells, which are known targets of Sirt2. Our findings demonstrate that miR-339 regulates Sirt2 in human and rat neurons. Since Sirt2 plays a critical role in multiple important cellular functions, our data imply that acupuncture may act through epigenetic changes and subsequent action on their targets, such as miRNA-339/Sirt2/NF-κB/FOXO1 axis. Some physiological level changes of neurons after altering the miR-339 levels are needed to validate the suggested therapeutic role of miR-339/Sirt2/NF-κB/FOXO1 axis in response to acupuncture therapy in the future work.
doi:10.1155/2015/249013
PMCID: PMC4324497
8.  A comprehensive database of Duchenne and Becker muscular dystrophy patients (0–18 years old) in East China 
Background
Currently, there is no cure for Duchenne and Becker muscular dystrophies (DMD/BMD). However, clinical trials with new therapeutic strategies are being conducted or considered. A comprehensive database is critical for patient recruitment and efficacy evaluation. China has the largest population, yet, no comprehensive database for DMD/BMD is available. Our study registered the data of the DMD/BMD patients in East China.
Methods
A modified registry form of Remudy (http://www.remudy.jp/) was applied to Chinese DMD/BMD patients through the outpatient clinic at Children’s Hospital of Fudan University, Shanghai during the period of August 2011 to December 2013. The data included geographic distribution of patients, age at diagnosis, clinical manifestation, genetic analysis and treatment status.
Results
194 DMD and 35 BMD patients were registered. Most patients lived in East China, namely Jiangsu province, Anhui province, Zhejiang province, Jiangxi province, Shanghai, Fujian province and Shandong province. All individuals aged less than 18 years (age limit to a children’s hospital). Diagnosis was made for a majority of patients during the age of 3–4 (16.6%) and 7–8 (14.8%) years old. Exon deletion was the most frequent genetic mutations (65.5% and 74.3%) followed by point mutations (14.4% and 11.4%), duplications (9.8% and 8.6%) and small insertion/deletion (9.3% and 2.9%) for DMD and BMD, respectively. 82.5% of DMD registrants were ambulatory, and all the BMD registrants were able to walk. 26.3% of DMD registrants have been treated with steroids. Cardiac functions were examined for 46.4% DMD boys and 45.7% BMD boys and respiratory functions were examined for 18.6% DMD boys and 14.3% BMD boys. Four boys with abnormal cardiac function were prescribed for treatment with cardiac medicine. 33.2% of DMD patients are eligible for exon skipping therapy, and among them 9.2% and 4.3% patients are eligible for skipping exon 51 and 53, respectively.
Conclusions
The database is the first linking accurate genetic diagnosis with clinical manifestation and treatment status of dystrophinopathy patients in East China. It provides comprehensive information essential for further patient management, especially for promotion of international cooperation in developing experimental therapies such as exon skipping and read-through of nonsense mutations targeting a subgroup of DMD patient population.
doi:10.1186/s13023-014-0220-7
PMCID: PMC4323212  PMID: 25612904
Duchenne and Becker muscular dystrophy; The CHFU database; Patient management
9.  Mono-isotope Prediction for Mass Spectra Using Bayes Network 
Tsinghua science and technology  2014;19(6):617-623.
Mass spectrometry is one of the widely utilized important methods to study protein functions and components. The challenge of mono-isotope pattern recognition from large scale protein mass spectral data needs computational algorithms and tools to speed up the analysis and improve the analytic results. We utilized naïve Bayes network as the classifier with the assumption that the selected features are independent to predict mono-isotope pattern from mass spectrometry. Mono-isotopes detected from validated theoretical spectra were used as prior information in the Bayes method. Three main features extracted from the dataset were employed as independent variables in our model. The application of the proposed algorithm to publicMo dataset demonstrates that our naïve Bayes classifier is advantageous over existing methods in both accuracy and sensitivity.
doi:10.1109/TST.2014.6961030
PMCID: PMC4302766  PMID: 25620856
Bayes network; tandem mass spectrum; mono-isotope prediction
10.  Photothermoelectric and photovoltaic effects both present in MoS2 
Scientific Reports  2015;5:7938.
As a finite-energy-bandgap alternative to graphene, semiconducting molybdenum disulfide (MoS2) has recently attracted extensive interest for energy and sensor applications. In particular for broad-spectral photodetectors, multilayer MoS2 is more appealing than its monolayer counterpart. However, little is understood regarding the physics underlying the photoresponse of multilayer MoS2. Here, we employ scanning photocurrent microscopy to identify the nature of photocurrent generated in multilayer MoS2 transistors. The generation and transport of photocurrent in multilayer MoS2 are found to differ from those in other low-dimensional materials that only contribute with either photovoltaic effect (PVE) or photothermoelectric effect (PTE). In multilayer MoS2, the PVE at the MoS2-metal interface dominates in the accumulation regime whereas the hot-carrier-assisted PTE prevails in the depletion regime. Besides, the anomalously large Seebeck coefficient observed in multilayer MoS2, which has also been reported by others, is caused by hot photo-excited carriers that are not in thermal equilibrium with the MoS2 lattice.
doi:10.1038/srep07938
PMCID: PMC4300500  PMID: 25605348
11.  The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family 
A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent “essential” plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.
doi:10.3389/fmicb.2014.00777
PMCID: PMC4290620  PMID: 25628616
broad-host-range plasmid; PromA plasmid family; complete sequence; plasmid backbone regions; comparative genomic analysis
12.  Genome Sequence of Thermoactinomyces daqus H-18, a Novel Thermophilic Species Isolated from High-Temperature Daqu 
Genome Announcements  2015;3(1):e01394-14.
Thermoactinomyces daqus H-18 is a new species of Thermoactinomyces isolated from high-temperature Daqu used in the fermentation of Bandongjing sesame-flavor liquor. Its genome was sequenced and assembled (3.44 Mb). The coding sequences (CDSs) that correlated to high-temperature tolerance were annotated. The metabolic pathways for the compounds responsible for flavor were also found.
doi:10.1128/genomeA.01394-14
PMCID: PMC4290991  PMID: 25573938
13.  Estradiol 17β and Its Metabolites Stimulate Cell Proliferation and Antagonize Ascorbic Acid-Suppressed Cell Proliferation in Human Ovarian Cancer Cells 
Reproductive Sciences  2014;21(1):102-111.
Estradiol 17β (E2β) and ascorbic acid (AA) have been implicated in cancer progression. However, little is known about the actions of biologically active metabolites of E2β, 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 2-methoxyestradiol (2ME2), and 4-methoxyestradiol (4ME2) synthesized sequentially by cytochrome P450, family 1, subfamily A (CYP1A1) and B (CYP1B1), polypeptide 1, and catechol-O-methyltransferase (COMT) on ovarian cancer. Herein, we examined the expression of CYP1A1, CYP1B1, COMT, and estrogen receptor α (ERα) and β (ERβ) in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, and OVCA-432). We also investigated the roles of E2β, 2OHE2, 4OHE2, 2ME2, and 4ME2 in cell proliferation, and their interactive effects with AA on ovarian cells. We found the expression of CYP1A1, CYP1B1, COMT, ERα, and ERβ in most cell lines tested. Treating cells with physiological concentrations of E2β and its metabolites promoted (13%-42% of the control) IOSE-385 and OVCAR-3 proliferation. The ER blockade inhibited IOSE-385 (∼76%) and OVCAR-3 (∼87%) proliferative response to E2β but not to its metabolites. The ERα blockade inhibited (∼85%) E2β-stimulated OVCAR-3 proliferation, whereas ERβ blockade attenuated (∼83%) E2β-stimulated IOSE-385 proliferation. The AA at ≥250 μmol/L completely inhibited serum-stimulated cell proliferation in all cell lines tested; however, such inhibition in IOSE-385, OVCAR-3, and OVCA-432 was partially (∼10%-20%) countered by E2β and its metabolites. Thus, our findings indicate that E2β and its metabolites promote cell proliferation and antagonize the AA-suppressed cell proliferation in a subset of ovarian cancer cells, suggesting that blocking the actions of E2β and its metabolites may enhance AA’s antiovarian cancer activity.
doi:10.1177/1933719113492211
PMCID: PMC3857769  PMID: 23757313
E2β; E2β metabolites; ascorbic acid; ovarian cancer cells; growth
14.  Toll-like receptor 9 is required for chronic stress-induced immune suppression 
Neuroimmunomodulation  2013;21(1):1-7.
Psychological and physical stress can suppress the immune system in both humans and animals. The mechanism by which stress affects immune responses, however, remains poorly defined. Toll-like receptors (TLRs) play a key role in modulating immune responses and cell survival. The mechanisms by which TLRs modulates chronic stress are largely unexplored. In current study, we revealed that a deficiency of TLR9 is resistant to chronic stress-induced lymphocyte apoptosis. In addition, TLR9 knockout (KO) mice significantly diminish the chronic stress-induced up-regulation of corticosterone levels. Notably, we found that lymphocytes from both TLR9 KO mice and wild type mice were similarly sensitive to corticosteroid-induced cell apoptosis. Moreover, we demonstrated that a deficiency of TLR9 blocks chronic stress-induced imbalance of Th1 and Th2 cytokine levels. Taken together, our findings reveal that TLR9 plays an essential role in chronic stress-induced immune suppression.
doi:10.1159/000354610
PMCID: PMC3896995  PMID: 24080854
TLR9; Chronic stress; Corticosteroid; Immune suppression; Apoptosis
15.  Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP) 
Journal of neurochemistry  2013;128(2):315-329.
Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders.
doi:10.1111/jnc.12463
PMCID: PMC3947313  PMID: 24117863
ERK; phosphorylation; phosphatase; synaptic plasticity; Striatal enriched tyrosine phosphatases (STEP); neurological disorders
16.  Potential of Computer-aided Diagnosis of High Spectral and Spatial (HiSS) MRI in the Classification of Breast Lesions 
Purpose:
To compare the performance of CADx analysis of pre-contrast HiSS MRI to that of clinical DCE-MRI in the diagnostic classification of breast lesions.
Materials and Methods:
Thirty-four malignant and seven benign lesions were scanned using 2D HiSS and clinical 4D DCE-MRI protocols. Lesions were automatically segmented. Morphological features were calculated for HiSS whereas both morphological and kinetic features were calculated for DCE-MRI. After stepwise feature selection, Bayesian artificial neural networks merged selected features, and ROC analysis evaluated the performance with leave-one-lesion-out validation.
Results:
AUC values of 0.92 ± 0.06 and 0.90 ± 0.05 were obtained using CADx on HiSS and DCE-MRI, respectively, in the task of classifying benign and malignant lesions. While we failed to show that the higher HiSS performance was significantly better than DCE-MRI, non-inferiority testing confirmed that HiSS was not worse than DCE-MRI.
Conclusion:
CADx of HiSS (without contrast) performed similarly to CADx on clinical DCE-MRI; thus, computerized analysis of HiSS may provide sufficient information for diagnostic classification. The results are clinically important for patients in whom contrast agent is contra-indicated. Even in the limited acquisition mode of 2D single slice HiSS, by using quantitative image analysis to extract characteristics from the HiSS images, similar performance levels were obtained as compared to those from current clinical 4D DCE-MRI. As HiSS acquisitions become possible in 3D, CADx methods can also be applied. Since HiSS and DCE-MRI are based on different contrast mechanisms, the use of the two protocols in combination may increase diagnostic accuracy.
doi:10.1002/jmri.24145
PMCID: PMC4143529  PMID: 24023011
High spectral and spatial resolution (HiSS) MRI; computer-aided diagnosis (CADx); breast cancer; dynamic contrast enhanced MRI (DCE-MRI); contrast-agent induced nephrotoxicity
17.  The Association of the MTHFR c.1625A>C Genetic Variant with the Risk of Congenital Heart Diseases in the Chinese 
The purpose of this study is to investigate the association of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with the risk of congenital heart diseases (CHD). The genotypes of the MTHFR genetic variant were determined by the polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing methods. Our data suggested that the allelic and genotypic frequencies of CHD patients were significantly different from non-CHD controls. The MTHFR c.1625A>C genetic variant was significantly associated with the increased risk of CHD (CC vs. AA: odds ratio [OR]=2.29, 95% confidence interval [CI] 1.15–4.53, p=0.016; C vs. A: OR=1.47, 95% CI 1.11–1.96, p=0.008). Results from this study indicate that the MTHFR c.1625A>C genetic variant influences the risk of CHD in the studied population.
doi:10.1089/gtmb.2014.0253
PMCID: PMC4278078  PMID: 25494855
18.  A Vps21 endocytic module regulates autophagy 
Molecular Biology of the Cell  2014;25(20):3166-3177.
Vps21 plays a role in autophagy in addition to its role in endocytosis. Individual deletions of members of the endocytic Vps21 module, including a GEF and four effectors, result in autophagy defects and accumulation of autophagosomal clusters. Therefore the endocytic Vps21 module regulates autophagy.
In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21∆ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21∆ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome—endocytosis and autophagy—converge through the Vps21 and Ypt7 GTPase modules.
doi:10.1091/mbc.E14-04-0917
PMCID: PMC4196867  PMID: 25143401
19.  Draft Genome Sequence of a New Bacillus cereus Strain tsu1 
Genome Announcements  2014;2(6):e01294-14.
This paper reports the draft genome sequence of a new Bacillus cereus strain tsu1, isolated on an agar-cellulose plate. The draft genome sequence is 5.81 Mb, revealing 5,673 coding sequences. It contains genes for cellulose-degradation and biosynthesis pathways of polyhydroxybutyrate (PHB) and 8 rRNA genes (5S, 16S, and 23S).
doi:10.1128/genomeA.01294-14
PMCID: PMC4271158  PMID: 25523768
20.  Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp 
PLoS ONE  2014;9(12):e115232.
Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.
doi:10.1371/journal.pone.0115232
PMCID: PMC4269435  PMID: 25517999
21.  A Novel Binocular Vision System for Wearable Devices 
We present a novel binocular imaging system for wearable devices incorporating the biology knowledge of the human eyes. Unlike the camera system in smartphones, two fish-eye lenses with a larger angle of view are used, the visual field of the new system is larger, and the central resolution of output images is higher. This design leads to more effective image acquisition, facilitating computer vision tasks such as target recognition, navigation and object tracking.
doi:10.1109/NEBEC.2014.6972996
PMCID: PMC4266940  PMID: 25525322
binocular vision; wearable device; fish-eye lens
22.  Arsenite-Induced Pseudo-Hypoxia Results in Loss of Anchorage-Dependent Growth in BEAS-2B Pulmonary Epithelial Cells 
PLoS ONE  2014;9(12):e114549.
Epidemiology studies have established a strong link between lung cancer and arsenic exposure. Currently, the role of disturbed cellular energy metabolism in carcinogenesis is a focus of scientific interest. Hypoxia inducible factor-1 alpha (HIF-1A) is a key regulator of energy metabolism, and it has been found to accumulate during arsenite exposure under oxygen-replete conditions. We modeled arsenic-exposed human pulmonary epithelial cells in vitro with BEAS-2B, a non-malignant lung epithelial cell line. Constant exposure to 1 µM arsenite (As) resulted in the early loss of anchorage-dependent growth, measured by soft agar colony formation, beginning at 6 weeks of exposure. This arsenite exposure resulted in HIF-1A accumulation and increased glycolysis, similar to the physiologic response to hypoxia, but in this case under oxygen-replete conditions. This “pseudo-hypoxia” response was necessary for the maximal acquisition of anchorage-independent growth in arsenite-exposed BEAS-2B. The HIF-1A accumulation and induction in glycolysis was sustained throughout a 52 week course of arsenite exposure in BEAS-2B. There was a time-dependent increase in anchorage-independent growth during the exposure to arsenite. When HIF-1A expression was stably suppressed, arsenite-induced glycolysis was abrogated, and the anchorage-independent growth was reduced. These findings establish that arsenite exerts a hypoxia-mimetic effect, which plays an important role in the subsequent gain of malignancy-associated phenotypes.
doi:10.1371/journal.pone.0114549
PMCID: PMC4267735  PMID: 25513814
23.  Intracellular complexes of the early-onset torsion dystonia-associated AAA+ ATPase TorsinA 
SpringerPlus  2014;3:743.
A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.
doi:10.1186/2193-1801-3-743
PMCID: PMC4320221
Early-onset dystonia, TorsinA; AAA+ ATPase; Protein association
24.  Efficacy of EGFR Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Patients with/without EGFR-Mutation: Evidence Based on Recent Phase III Randomized Trials 
Background
EGFR mutation might be a predictive factor for applying EGFR-tyrosine kinase inhibitors (EGFR-TKIs, including gefitinib, erlotinib and afatinib) in non-small-cell lung cancer (NSCLS) patients. Thus, it is necessary to pool previous trials to compare the effect of EGFR-TKIs versus cytotoxic chemotherapy in EGFR mutation positive (mut+) and negative (mut−) patients.
Material/Methods
This study identified 8 first-line and 9 second-line phase III trials in databases. Hazard ratio (HR) was pooled to assess the risk of progression-free survival (PFS), and overall survival (OS), while odds ratio (OR) was pooled to assess objective response, disease control, and toxicity of EGFR-TKIs verses chemotherapy.
Results
In EGFR mut+ patients, EGFR-TKIs were associated with significantly lower risk of disease progression in the first-line setting, but this trend was only observed in the gefitinib group, not in the erlotinib group in the second-line setting. In EGFR mut− patients, gefitinib and erlotinib had significantly higher risk of disease progression in first-line and second-line setting, respectively. Compared with chemotherapy, the effects of EGFR-TKIs on OS in both first-line and second-line settings were not evident. Regarding toxicity, EGFR-TKIs had significantly higher risk of rash and lower hematological toxicity compared with chemotherapy.
Conclusions
All of the 3 EGFR-TKIs and gefitinib alone regimens had better effects in prolonging PFS in EGFR mut+ patients in first-line and second-line setting, respectively, but chemotherapy seemed more effective in EGFR mut− patients than EGFR-TKIs. Therefore, accurate identification of EGFR mutation status is useful to decide on an appropriate regimen for treatment of NSCLC patients.
doi:10.12659/MSM.892476
PMCID: PMC4271800  PMID: 25503781
Carcinoma, Non-Small-Cell Lung; Genes, erbB-1; Meta-Analysis
25.  Detection of JAK2 V617F mutation increases the diagnosis of myeloproliferative neoplasms 
Oncology Letters  2014;9(2):735-738.
The Janus kinase (JAK)2 gene, which is located on chromosome 9p24, is involved in the signaling transduction pathways of the hematopoietic and immune system. Mutations in the JAK2 gene have served as disease markers for myeloproliferative neoplasms (MPNs). The aim of the present study was to investigate the occurrence of the JAK2 gene mutation in 140 clinical samples, and to evaluate its clinical significance in MPNs and other hematological diseases. Genomic DNA was extracted from the peripheral blood leukocytes or bone marrow karyocytes of 140 clinical samples, which included 130 patients with various types of hematological disease and 10 control patients. In addition, exons 12 and 14 of the JAK2 gene were analyzed by direct sequencing and the mutation rates of various MPN subtypes were evaluated. Of the 140 samples, exons 12 and 14 were tested in 74 samples, however, exon 14 only was tested in 66 samples. No mutations were identified in exon 12. The V617F mutation rate in polycythemia vera was 82.1% (23/28), and the mutation rates in essential thrombocythemia histiocytosis, primary myelofibrosis and other MPNs were 53.1% (17/32), 40.0% (4/10) and 60.0% (6/10), respectively. Therefore, the total mutation rate of the JAK2 gene in MPN was 62.5% (50/80). For non-MPN hematological diseases, four V617F mutations were detected in samples of leukocytosis of unknown origin (4/12), however, no JAK2 V617F mutations were identified in the 10 controls. Therefore, JAK2 V617F mutations may present a novel marker for diagnosis of MPNs. Furthermore, the direct sequencing method appeared to be satisfactory for the clinical gene testing of hematological samples.
doi:10.3892/ol.2014.2801
PMCID: PMC4301535  PMID: 25624900
Janus kinase 2 gene; V617F; gene test; direct sequencing; myeloproliferative neoplasms

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