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1.  Salvage of Failed Protein Targets by Reductive Alkylation 
The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins.
PMCID: PMC4078742  PMID: 24590719
Chemical modification; Lysine reductive alkylation; Methylation; Ethylation; Isopropylation; Protein crystallization
2.  Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions 
Nature methods  2009;6(8):585-587.
We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we release the intact complex largely devoid of detergent This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.
PMCID: PMC4066579  PMID: 19578383
3.  Impact of Ketamine on Learning and Memory Function, Neuronal Apoptosis and Its Potential Association with miR-214 and PTEN in Adolescent Rats 
PLoS ONE  2014;9(6):e99855.
Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is used as a general pediatric anesthetic and anti-depressive drug. Recent studies suggest that ketamine enhances neuronal apoptosis in developing rats. The goal of this study is to explore whether ketamine could result in learning and memory impairment and neurodegeneration in adolescent rats, and if so, whether the effects of ketamine are associated with miR-214 and PTEN expression. Fifty-day-old SD rats were randomly divided into three groups receiving ketamine at 30, or 80 mg/kg, i.p. or saline for seven consecutive days. Twenty-four hours after the last treatment, learning and memory function were tested by the Morris water maze. The rats were then decapitated, and the brains were isolated for detection of neuronal apoptosis and protein PTEN expression by TUNEL and immunohistochemistry respectively. Expression levels of the miR-214 and PTEN in the hippocampus were measured by qRT-PCR and western blot analysis respectively. Ketamine administered to the adolescent rats at a dose of 80 mg/kg rather than the lower dose of 30 mg/kg caused learning and memory impairment, increased the number of apoptotic cells in the hippocampal CA1 region, cerebral cortex and subcortical region, decreased the miR-214 levels and increased PTEN protein expression in hippocampus. The results suggest that ketamine at a dose of 80 mg/kg in the adolescent rats is able to induce the learning and memory impairment and neurodegeneration, in which the down-regulation of miR-214 and high expression of PTEN protein may be involved.
PMCID: PMC4051782  PMID: 24914689
4.  Increased Cystic Fibrosis Transmembrane Conductance Regulators Expression and Decreased Epithelial Sodium Channel Alpha Subunits Expression in Early Abortion: Findings from a Mouse Model and Clinical Cases of Abortion 
PLoS ONE  2014;9(6):e99521.
The status of the maternal endometrium is vital in regulating humoral homeostasis and for ensuring embryo implantation. Cystic fibrosis transmembrane conductance regulators (CFTR) and epithelial sodium channel alpha subunits (ENaC-α) play an important role in female reproduction by maintaining humoral and cell homeostasis. However, it is not clear whether the expression levels of CFTR and ENaC-α in the decidual component during early pregnancy are related with early miscarriage. CBA×DBA/2 mouse mating has been widely accepted as a classical model of early miscarriage. The abortion rate associated with this mating was 33.33% in our study. The decidua of abortion-prone CBA female mice (DBA/2 mated) had higher CFTR mRNA and protein expression and lower ENaC-α mRNA and protein expression, compared to normal pregnant CBA mice (BLAB/C mated). Furthermore, increased CFTR expression and decreased ENaC-α expression were observed in the uterine tissue from women with early miscarriage, as compared to those with successful pregnancy. In conclusion, increased CFTR expression and decreased ENaC-α expression in the decidua of early abortion may relate with failure of early pregnancy.
PMCID: PMC4051784  PMID: 24914548
5.  Pharmacokinetics, clearance, and biosafety of polyethylene glycol-coated hollow gold nanospheres 
Gold nanoparticles have attracted enormous interest as potential theranostic agents. However, little is known about the long-term elimination and systemic toxicity of gold nanoparticles in the literature. Hollow gold nanospheres (HAuNS) is a class of photothermal conducting agent that have shown promises in photoacoustic imaging, photothermal ablation therapy, and drug delivery. It’s very necessary to make clear the biosafety of HAuNS for its further application.
We investigated the cytotoxicity, complement activation, and platelet aggregation of polyethylene glycol (PEG)-coated HAuNS (PEG-HAuNS, average diameter of 63 nm) in vitro and their pharmacokinetics, biodistribution, organ elimination, hematology, clinical chemistry, acute toxicity, and chronic toxicity in mice.
PEG-HAuNS did not induce detectable activation of the complement system and did not induce detectable platelet aggregation. The blood half-life of PEG-HAuNS in mice was 8.19 ± 1.4 hr. The single effective dose of PEG-HAuNS in photothermal ablation therapy was determined to be 12.5 mg/kg. PEG-HAuNS caused no adverse effects after 10 daily intravenous injections over a 2-week period at a dose of 12.5 mg/kg per injection (accumulated dose: 125 mg/kg). Quantitative analysis of the muscle, liver, spleen, and kidney revealed that the levels of Au decreased 45.2%, 28.6%, 41.7%, and 40.8%, respectively, from day 14 to day 90 after the first intravenous injection, indicating that PEG-HAuNS was slowly cleared from these organs in mice.
Our data support the use of PEG-HAuNS as a promising photothermal conducting agent.
PMCID: PMC4082425  PMID: 24886070
Hollow gold nanospheres; Toxicity; Photothermal ablation therapy
6.  Epidemiology, species distribution and outcome of nosocomial Candida spp. bloodstream infection in Shanghai 
BMC Infectious Diseases  2014;14:241.
Yeasts, mostly Candida, are important causes of bloodstream infections (BSI), responsible for significant mortality and morbidity among hospitalized patients. The epidemiology and species distribution vary from different regions. The goals of this study were to report the current epidemiology of Candida BSI in a Shanghai Teaching Hospital and estimate the impact of appropriate antifungal therapy on the outcome.
From January 2008 to December 2012, all consecutive patients who developed Candida BSI at Ruijin University Hospital were enrolled. Underlying diseases, clinical severity, species distribution, antifungal therapy and its impact on the outcome were analyzed.
A total of 121 episodes of Candida BSI were identified, with an incidence of 0.32 episodes/1,000 admissions (0.21 in 2008 and 0.42 in 2012) The proportion of candidemia caused by non-albicans species (62.8%), including C. parapsilosis (19.8%), C. tropicalis (14.9%), C. glabrata (7.4%), C. guilliermondii (5.8%), C. sake (5.0%) was higher than that of candidemia caused by C. albicans (37.2%). The overall crude 28-day mortality was 28.1% and significantly reduced with appropriate empiric antifungal therapy administered within 5 days (P = 0.006). Advanced age (OR 1.04; P = 0.014), neutropenia < 500/mm3 (OR 17.44; P < 0.001) were independent risk factors for 28-day mortality, while appropriate empiric antifungal therapy (OR 0.369; P = 0.035) was protective against 28-day mortality.
The epidemiology of candidemia in Shanghai differed from that observed in Western countries. Appropriate empiric antifungal therapy influenced the short-term survival.
PMCID: PMC4033490  PMID: 24886130
Candida spp; Bloodstream infection; Appropriate antifungal therapy; Survival
7.  Annexin A5-Functionalized Nanoparticle for Multimodal Imaging of Cell Death 
Molecular imaging  2013;12(3):182-190.
Techniques for visualizing cell death can provide noninvasive assessment of both disease states and response to therapeutic intervention. The purpose of this study was to develop and evaluate a multimodal imaging nanoplatform for the detection of cell death.
In this study, we evaluated 111In-labeled Annexin A5-conjugated core-crosslinked polymeric micelles (CCPM) for multimodal imaging of cell death in various disease models. Three different models were conducted, including tumor apoptosis, hepatic apoptosis and inflammation. Both micro-single photon emission tomography/computed tomography (µSPECT/CT) and fluorescence molecular tomography (FMT) were performed. Biodistribution and immunohistochemistry assays were carried out to validate selectivity of cell death imaging.
In all disease models, cell death was clearly visualized by both µSPECT/CT and FMT. In contrast, there was relatively low signal in the corresponding tissues of control mice. Moreover, the radioactive signal from 111In-labeled annexin A5-CCPM colocalized with its fluorescence signal, and both signals were confined to regions of dying cells.
111In-labeled annexin A5-CCPM allows visualization of cell death by both nuclear and optical techniques at whole-body level as well as at microscopic level. It has the potential to aid diagnosis of disease states or tissue responses involving abnormal cell death.
PMCID: PMC3893065  PMID: 23490444
Cell Death; Annexin A5; Polymeric Micelles; Nuclear Imaging; Fluorescence Optical Imaging
8.  The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture 
The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance.
The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.
PMCID: PMC3975887  PMID: 24699643
glutamate-ammonia ligase; leguminous; herbicide resistance; Medicago truncatula
9.  Dual patch voltage clamp study of low membrane resistance astrocytes in situ 
Molecular Brain  2014;7:18.
Whole-cell patch clamp recording has been successfully used in identifying the voltage-dependent gating and conductance properties of ion channels in a variety of cells. However, this powerful technique is of limited value in studying low membrane resistance cells, such as astrocytes in situ, because of the inability to control or accurately measure the real amplitude of command voltages. To facilitate the study of ionic conductances of astrocytes, we have developed a dual patch recording method which permits membrane current and membrane potential to be simultaneously recorded from astrocytes in spite of their extraordinarily low membrane resistance. The utility of this technique is demonstrated by measuring the voltage-dependent activation of the inwardly rectifying K+ current abundantly expressed in astrocytes and multiple ionic events associated with astrocytic GABAA receptor activation. This protocol can be performed routinely in the study of astrocytes. This method will be valuable for identifying and characterizing the individual ion channels that orchestrate the electrical activity of low membrane resistance cells.
PMCID: PMC3995526  PMID: 24636341
Astrocytes; Voltage clamp; Membrane resistance; Kir4.1; GABAA receptor
10.  Preventive effects of basic fibroblast growth factor on vascular restenosis after balloon angioplasty 
The aim of the present study was to investigate whether chronic administration of basic fibroblast growth factor (bFGF) following angioplasty in a dog model of atherosclerotic iliac stenosis may restore endothelium function and prevent restenosis (RS). In total, 40 dogs with atherosclerotic stenosis of the right iliac arteries were used in the study. A total of 20 dogs underwent histological examination of the lumen areas prior to (n=10) and immediately following angioplasty (n=10). Intravenous bFGF was administered to 10 dogs (bFGF group) and an additional 10 dogs received vehicle injection (control group). Animals in the two groups were sacrificed 42 days following surgery for in vitro analysis of vascular reactivity and morphometric assessment of the histological cross-sectional areas. The bFGF group exhibited significantly greater maximal endothelium-dependent acetylcholine-induced relaxation (Emax, 43±9%) when compared with the control group (Emax, 8±6%; P<0.05). In addition, the maximal endothelium-independent response of the bFGF group to sodium nitroprusside (Emax, 90±2%) was greater than that of the control group (Emax, 60±2%; P<0.05). Six weeks following angioplasty, the lumen area in the bFGF group (2.01±0.78 mm2) was greater compared with the control group (1.0±0.10%). The lumen area decreased by 58% between immediately after angioplasty and the control group six weeks following angioplasty. Therefore, the results of the present study indicated that administration of bFGF may not only restore endothelium-dependent and -independent relaxation, but also prevent RS in dogs that have undergone angioplasty.
PMCID: PMC3991498  PMID: 24940410
basic fibroblast growth factor; angioplasty; vascular endothelium
11.  Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding 
Science (New York, N.Y.)  2011;334(6054):380-385.
The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalised by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane embedded molecular machines in the gas phase. Here we show that rotary ATPases/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently we can link specific lipid and nucleotide binding with distinct regulatory roles.
PMCID: PMC3927129  PMID: 22021858
12.  Lack of Association between Insulin Receptor Substrate2 rs1805097 Polymorphism and the Risk of Colorectal and Breast Cancer: A Meta-Analysis 
PLoS ONE  2014;9(1):e86911.
Insulin receptor substrate-2 (IRS-2), a signaling adaptor protein, was involved in two cancer-related pathways (the phosphatidylinositol 3′-kinase (PI3K) and the extracellular signal-regulated kinase (ERK) pathways). Several studies have evaluated the association between IRS2 rs1805097 (G>A) polymorphisms and the risk of colorectal and breast cancer. However, the results were inconsistent.
Methodology/Principal Findings
A meta-analysis of seven published case-control studies (4 studies with 4798 cases and 5478 controls for colorectal cancer and 3 studies with 2108 cases and 2507 controls for breast cancer) were conducted to assess the strength of association using crude odd ratios (ORs) with 95% confidence intervals (CIs). For colorectal cancer, no obvious associations were found for all genetic models (homozygote comparison OR = 0.96, 95%CI 0.85–1.08, Pheterogeneity = 0.97; heterozygote comparison: OR = 0.91, 95%CI 0.73–1.13, Pheterogeneity<0.01; dominant model: OR = 0.92, 95%CI 0.80–1.06, Pheterogeneity = 0.05; recessive model: OR = 1.02, 95%CI 0.91–1.14, Pheterogeneity = 0.60). In the subgroup analysis by ethnicity, control source and consistency of frequency with Hardy-Weinberg equilibrium (HWE), still no significant associations were observed. For breast cancer, also no obvious associations were found for all genetic models (homozygote comparison: OR = 0.95, 95%CI 0.71–1.26, Pheterogeneity = 0.10; heterozygote comparison: OR = 1.00, 95%CI 0.89–1.14, Pheterogeneity = 0.71; dominant model: OR = 0.98, 95%CI 0.87–1.10, Pheterogeneity = 0.55; recessive model: OR = 0.95, 95%CI 0.72–1.25, Pheterogeneity = 0.07). We performed subgroup analyses by sample size and did not find an association.
This meta-analysis indicated that IRS2 rs1805097polymorphism was not associated with colorectal and breast cancer risk.
PMCID: PMC3907441  PMID: 24497996
13.  Bicarbonate efflux via GABAA receptors depolarizes membrane potential and inhibits two-pore domain potassium channels of astrocytes in rat hippocampal slices 
Glia  2012;60(11):1761-1772.
Increasing evidence indicates the functional expression of ionotropic γ-aminobutyric acid receptor (GABAA-R) in astrocytes. However, it remains controversial in regard to the intracellular Cl− concentration ([Cl−]i) and the functional role of anion-selective GABAA-R in astrocytes. In gramicidin perforated-patch recordings from rat hippocampal CA1 astrocytes, GABA and GABAA-R specific agonist THIP depolarized astrocyte membrane potential (Vm), and the THIP induced currents reversed at the voltages between −75.3 to −78.3 mV, corresponding to a [Cl−]i of 3.1 – 3.9 mM that favors a passive distribution of Cl− anions across astrocyte membrane. Further analysis showed that GABAA-R induced Vm depolarization is ascribed to HCO3− efflux, while a passively distributed Cl− mediates no net flux or influx of Cl-that leads to an unchanged or hyperpolarized Vm. In addition to a rapidly activated GABAA-R current component, GABA and THIP also induced a delayed inward current (DIC) in 63% of astrocytes. The DIC became manifest after agonist withdrawal and enhanced in amplitude with increasing agonist application duration or concentrations. Astrocytic two-pore domain K+ channels (K2Ps), especially TWIK-1, appeared to underlie the DIC, because 1) acidic intracellular pH, as a result of HCO3− efflux, inhibited TWIK-1; 2) the DIC remained in the Cs+ recording solutions that inhibited conventional K+ channels and 3) the DIC was completely inhibited by 1 mM quinine but not by blockers for other cation/anion channels. Altogether, HCO3− efflux through activated GABAA-R depolarizes astrocyte Vm and induces a delayed inhibition of K2Ps K+ channels via intracellular acidification.
PMCID: PMC3901573  PMID: 22855415
Astrocytes; GABAA receptors; bicarbonate; TWIK-1; patch clamp; hippocampus
14.  Human Parainfluenza Virus Type 2 V Protein Inhibits TRAF6-Mediated Ubiquitination of IRF7 To Prevent TLR7- and TLR9-Dependent Interferon Induction 
Journal of Virology  2013;87(14):7966-7976.
Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.
PMCID: PMC3700191  PMID: 23678181
15.  EGFRvIII Mediates Hepatocellular Carcinoma Cell Invasion by Promoting S100 Calcium Binding Protein A11 Expression 
PLoS ONE  2013;8(12):e83332.
Epidermal growth factor receptor (EGFR) is frequently aberrantly expressed in cancer, and abnormal signalling downstream of this receptor contributes to tumour growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. Aberrant signalling downstream of this receptor contributes to tumour invasion. We previously reported that EGFRvIII can promote hepatocellular carcinoma (HCC) invasion. However, little is known concerning the mechanisms underlying EGFRvIII-mediated increases in cell motility and invasion in HCC. In this study, we observed that S100A11 was significantly upregulated in Huh-7 cells that overexpressed EGFRvIII. Moreover, S100A11 expression was elevated in HCC tissue samples (68.6%; 35/51), and this elevation was correlated with EGFRvIII expression (p = 0.0020; n = 20). Furthermore, the overexpression of S100A11 can promote HCC cell invasiveness, whereas siRNA against S100A11 can suppress the invasiveness of HCC cells stably transfected with EGFRvIII. Additionally, STAT3 inhibitors can block S100A11 expression and S100A11 promoter activity in HCC cells with stable overexpression of EGFRvIII. Furthermore, mutation in STATx binding sites could abolish the S1000A11 promoter activity stimulation by EGFRvIII. Taken together, the results demonstrate that the EGFRvIII-STAT3 pathway promotes cell migration and invasion by upregulating S100A11.
PMCID: PMC3869758  PMID: 24376686
16.  The Prognostic Value of Phosphorylated AKT Expression in Non-Small Cell Lung Cancer: A Meta-Analysis 
PLoS ONE  2013;8(12):e81451.
Phosphorylated AKT (p-AKT), constitutive activation of AKT, is a potentially interesting prognostic marker and therapeutic target in non-small cell lung cancer (NSCLC). However, the available results of p-AKT expression in NSCLC are heterogeneous. Therefore, a meta-analysis of published researches investigating the prognostic relevance of p-AKT expression in patients with NSCLC was performed.
Materials and Methods
A literature search via PubMed, EMBASE and CNKI (China National Knowledge Infrastructure) databases was conducted. Data from eligible studies were extracted and included into meta-analysis using a random effects model.
A total of 1049 patients from nine studies were included in the meta-analysis. Nine studies investigated the relationship between p-AKT expression and overall survival using univariate analysis, and five of these undertook multivariate analysis. The pooled hazard ratio (HR) for overall survival was 1.49 (95% confidence interval (CI): 1.01-2.20) by univariate analysis and 1.02 (95% CI: 0.54-1.95) by multivariate analysis.
Our study shows that positive expression of p-AKT is associated with poor prognosis in patients with NSCLC. However, adequately designed prospective studies need to perform.
PMCID: PMC3857807  PMID: 24349071
17.  The crystal structures of the α-subunit of the α2β2 tetrameric Glycyl-tRNA synthetase 
Aminoacyl-tRNA synthetases (AARSs) are ligases (EC.6.1.1.-) that catalyze the acylation of amino acids to their cognate tRNAs in the process of translating genetic information from mRNA to protein. Their amino acid and tRNA specificity are crucial for correctly translating the genetic code. Glycine is the smallest amino acid and the glycyl-tRNA synthetase (GlyRS) belongs to Class II AARSs. The enzyme is unusual because it can assume different quaternary structures. In eukaryotes, archaebac-teria and some bacteria, it forms an α2 homodimer. In some bacteria, GlyRS is an α2β2 heterotetramer and shows a distant similarity to α2 GlyRSs. The human pathogen eubacterium Campylobacter jejuni GlyRS (CjGlyRS) is an α2β2 heterotetramer and is similar to Escherichia coli GlyRS; both are members of Class IIc AARSs. The two-step aminoacylation reaction of tetrameric GlyRSs requires the involvement of both α- and β-subunits. At present, the structure of the GlyRS α2β2 class and the details of the enzymatic mechanism of this enzyme remain unknown. Here we report the crystal structures of the catalytic α-subunit of CjGlyRS and its complexes with ATP, and ATP and glycine. These structures provide detailed information on substrate binding and show evidence for a proposed mechanism for amino acid activation and the formation of the glycyl-adenylate intermediate for Class II AARSs.
PMCID: PMC3691008  PMID: 23054484
Gly-tRNA synthetase; Catalytic subunit; ATP binding; Glycine binding
18.  Effects of integrin-targeted photodynamic therapy on pancreatic carcinoma cell 
AIM: To investigate the effects of photodynamic therapy with quantum dots-arginine-glycine-aspartic acid (RGD) probe as photosensitizer on the proliferation and apoptosis of pancreatic carcinoma cells.
METHODS: Construction of quantum dots-RGD probe as photosensitizer for integrin-targeted photodynamic therapy was accomplished. After cells were treated with photodynamic therapy (PDT), the proliferation of SW1990 cells were measured by methyl thiazolyl tetrazolium assay. Morphologic changes, cell cycle retardance and apoptosis were observed under fluoroscope and flow cytometry. The expression of myeloid cell leukemia-1 (Mcl-1), protein kinase B (Akt) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA were detected by reverse transcription-polymerase chain reaction. The amount of reactive oxygen species were also evaluated by fluorescence probe.
RESULTS: The photodynamic therapy with quantum dots-RGD probe as photosensitizer significantly inhibited cell proliferation (P < 0.01). Apoptotic cells and morphologic changes could be found under optical microscope. The FCM revealed PDT group had more significant cell apoptosis rate compared to control cells (F = 130.617, P < 0.01) and cell cycle G0/G1 and S retardance (P < 0.05) compared to control cells. The expression of Mcl-1 and Akt mRNA were down-regulated, while expression of TRAIL mRNA was up-regulated after cells treated with PDT. PDT group had more significant number of cells producing reactive oxygen species compared to control cells (F = 3262.559, P < 0.01).
CONCLUSION: The photodynamic therapy with quantum dots-RGD probe as photosensitizer significantly inhibits cell proliferation and increases apoptosis in SW1990 cells.
PMCID: PMC3801369  PMID: 24151382
Pancreatic carcinoma; Targeted probe; Photodynamic therapy; Apoptosis; Reactive oxygen species
19.  Structure of the CRISPR Interference Complex CSM Reveals Key Similarities with Cascade 
Molecular Cell  2013;52(1):124-134.
The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I–III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family.
Graphical Abstract
•The CSM complex from Sulfolobus solfataricus has been purified and characterized•EM reveals a helical backbone with striking similarities to the Cascade complex•Mass spectrometry defines the subunit stoichiometry and organization of the complex•CSM subunits are modified by methylation, acetylation, and phosphorylation
PMCID: PMC3807668  PMID: 24119402
20.  Copper Sulfide Nanoparticles as a New Class of Photoacoustic Contrast Agent for Deep Tissue Imaging at 1064-nm 
ACS nano  2012;6(8):7489-7496.
Photoacoustic tomography (PAT) is an emerging molecular imaging modality. Here, we demonstrate use of semiconductor copper sulfide nanoparticles (CuS NP) for PAT with an Nd:YAG laser at a wavelength of 1064 nm. CuS NP allowed visualization of mouse brain after intracranial injection, rat lymph nodes 12 mm below the skin after interstitial injection, and CuS NP-containing agarose gel embedded in chicken breast muscle at the depth of ~ 5 cm. This imaging approach has great potential for molecular imaging of breast cancer.
PMCID: PMC3429766  PMID: 22812694
photoacoustic tomography; CuS nanoparticles; 1064-nm laser; optical imaging
21.  Genetic polymorphisms of XPD and CDA and lung cancer risk 
Oncology Letters  2012;4(2):247-251.
To determine the susceptibility genes of lung cancer, we investigated the frequency distributions of the xeroderma pigmentosum complementary group D (XPD) and cytidine deaminase (CDA) genes in patients. A case-control study was conducted involving lung cancer patients and healthy controls. The genotypic distributions of XPD exon 10 G→A (Asp312Asn) and 23 T→G (Lys751Gln), and CDA 79 A→C (Lys27Gln) and 208 G→A (Ala70Thr), were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results demonstrated that the XPD Asp312Asn genotype distribution was G/G (82.52%) and A/G (17.48%) in the lung cancer patients, and G/G (82.52%), A/G (16.50%) and A/A (10.98%) in the controls. The genotypes of Lys751Gln were T/T (83.49%) and T/G (16.50%) in the lung cancer patients, and T/T (84.47%) and T/G (15.53%) in the controls. Mutations in the XPD single nucleotide polymorphism loci did not demonstrate a significant difference between the two groups (P>0.05). The risk of lung cancer in individuals with mutations at positions 312 and 751 increased 6.13-fold (P=0.047). The CDA Lys27Gln genotype distribution was A/A (78.65%), A/C (20.39%) and C/C (0.98%) in the lung cancer patients, and A/A (79.61%), A/C (19.42%) and C/C (0.98%) in the controls (P=0.985). The CDA Ala70Thr genotype distribution was G/G (98.06%) and A/G (1.94%) in the controls, while all the genotypes were wild-type in the lung cancer patients. The difference between the lung cancer patients and the controls was not statistically significant (P=0.155). There was also no significant difference in the frequency distribution of XPD or CDA between the different pathological types (P>0.05). Our findings demonstrate that the mutation of XPD codons 312 and 751 increases the risk of lung cancer. By contrast, polymorphisms of CDA appear to have little association with lung cancer.
PMCID: PMC3402721  PMID: 22844363
genetic polymorphisms; xeroderma pigmentosum complementary group D; cytidine deaminase; lung cancer
22.  Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis 
Journal of molecular biology  2012;420(0):366-383.
Microcin C (McC) is heptapeptide-adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine-peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its AMP-complex reveal specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.
PMCID: PMC3690760  PMID: 22516613
MccF; serine peptidase; nucleophilic elbow; catalytic triad (Ser-His-Glu); substrate binding loop
23.  Global Identification of Peptidase Specificity by Multiplex Substrate Profiling 
Nature methods  2012;9(11):1095-1100.
A simple and rapid multiplex substrate profiling method has been developed to reveal the substrate specificity of any endo- or exo-peptidase using LC-MS/MS sequencing. A physicochemically diverse library of peptides was generated by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated prime and non-prime site information and substrate specificity matched or expanded upon previous substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C gene responsible for polypeptide processing and allowed Granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor ion abundance. Furthermore, the proteolytic secretions from a parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.
PMCID: PMC3707110  PMID: 23023596
24.  Low Dose Decitabine Treatment Induces CD80 Expression in Cancer Cells and Stimulates Tumor Specific Cytotoxic T Lymphocyte Responses 
PLoS ONE  2013;8(5):e62924.
Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8+, but not CD4+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.
PMCID: PMC3650049  PMID: 23671644
25.  Effects of medical adhesives in prevention of complications after endoscopic submucosal dissection 
AIM: To evaluate the use of medical adhesive spray in endoscopic submucosal dissection (ESD).
METHODS: Patients who underwent ESD between January 2009 and June 2012 (n = 173) were enrolled in the prospective randomized study. Two patients undergoing surgery due to severe intraoperative hemorrhage and failed hemostasis were excluded, and the remaining 171 patients were randomly divided into two groups: group A (medical adhesive group, n = 89) and group B (control group, n = 82). In group A, a medical adhesive spray was evenly applied after routine electrocoagulation and hemostasis using hemostatic clip after ESD. Patients in group B only treated with routine wound management. Intraoperative and postoperative data were collected and compared.
RESULTS: In all 171 patients, ESD was successfully completed. There was no significant difference in the average treatment time between groups A and B (59.4 min vs 55.0 min, respectively). The average length of hospital stay was significantly different between group A and B (8.89 d vs 9.90 d, respectively). The incidence of intraoperative perforation was 10.1% in group A and 9.8% in group B, and was not significantly different between the two groups. In all cases, perforations were successfully managed endoscopically and with conservative treatment. The incidence of postoperative delayed bleeding in group A was significantly lower than that in group B (0.00% vs 4.88%, respectively).
CONCLUSION: ESD is an effective minimally invasive treatment for gastrointestinal precancerous lesions or early-stage gastrointestinal cancer. Medical adhesive spray is effective in preventing delayed bleeding after ESD, and can thus reduce the average length of hospital stay.
PMCID: PMC3645390  PMID: 23674879
Endoscopic submucosal dissection; Medical adhesive; Early-stage gastrointestinal cancer; Postoperative delayed bleeding; Intraoperative hemorrhage

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