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1.  4-[(Z)-(2-Fur­yl)(2-naphthyl­amino)methyl­ene)]-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one. Corrigendum 
Corrigendum to Acta Cryst. (2009), E65, o1824.
The title of the paper by Li, Li, Li, Zhang & Li [Acta Cryst. (2009), E65, o1824] is corrected.
doi:10.1107/S1600536810002527
PMCID: PMC2979883  PMID: 21579582
2.  4-[(Z)-(2-Fur­yl)(2-naphthyl­amino)methyl­ene)]-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one 
The title compound, C25H19N3O2, crystallizes as discrete mol­ecules which are well ordered through one intra­molecular N—H⋯O hydrogen bond. Structural analysis indicates that the mol­ecules exist as the amine–one form.
doi:10.1107/S1600536809025586
PMCID: PMC2977223  PMID: 21583526
3.  Complement Activation and Intraventricular Rituximab Distribution in Recurrent Central Nervous System Lymphoma 
Purpose
To elucidate the mechanistic basis for efficacy of intrathecal rituximab. We evaluated complement activation as well as the pharmacokinetics of intraventricular rituximab in patients who participated in two phase 1 multicenter studies.
Experimental Design
We evaluated complement activation as a candidate mediator of rituximab within the CNS. Complement C3 and C5b-9 were quantified by ELISA in serial CSF specimens after intraventricular rituximab administration. We determined rituximab concentration profiles in CSF and serum. A population three- compartment pharmacokinetic model was built to describe the disposition of rituximab following intraventricular administration. The model was derived from results of the first trial and validated with results of the second trial.
Results
Complement C3 and C5b-9 were reproducibly activated in CSF after intraventricular rituximab. Ectopic expression of C3 mRNA and protein within CNS lymphoma lesions was localized to myeloid cells. Constitutive high C3 activation at baseline was associated with adverse prognosis. A PK model was built which contains three distinct compartments to describe the distribution of rituximab within the neuroaxis after intraventricular administration.
Conclusions
We provide the first evidence of C3 activation within the neuroaxis with intraventricular immunotherapy and suggest that complement may contribute to immunotherapeutic responses of rituximab in CNS lymphoma. Penetration of rituximab into neural tissue is supported by this pharmacokinetic model and may contribute to efficacy. These findings have general implications for intraventricular immunotherapy. Our data highlight potential innovations to improve efficacy of intraventricular immunotherapy both via modulation of the innate immune response as well as innovations in drug delivery.
doi:10.1158/1078-0432.CCR-13-0474
PMCID: PMC3944388  PMID: 24190981
Complement; Immunotherapy; Non-Hodgkin’s Lymphoma; Rituximab; Pharmacokinetics
4.  Aurora B-dependent phosphorylation of Ataxin-10 promotes the interaction between Ataxin-10 and Plk1 in cytokinesis 
Scientific Reports  2015;5:8360.
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder caused by ATTCT expansion in the ATXN10 gene. Previous investigations have identified that depletion of Ataxin-10, the gene product, leads to cellular apoptosis and cytokinesis failure. Herein we identify the mitotic kinase Aurora B as an Ataxin-10 interacting partner. Aurora B interacts with and phosphorylates Ataxin-10 at S12, as evidenced by in vitro kinase and mass spectrometry analysis. Both endogenous and S12-phosphorylated Ataxin-10 localizes to the midbody during cytokinesis, and cytokinetic defects induced by inhibition of ATXN10 expression is not rescued by the S12A mutant. Inhibition of Aurora B or expression of the S12A mutant renders reduced interaction between Ataxin-10 and polo-like kinase 1 (Plk1), a kinase previously identified to regulate Ataxin-10 in cytokinesis. Taken together, we propose a model that Aurora B phosphorylates Ataxin-10 at S12 to promote the interaction between Ataxin-10 and Plk1 in cytokinesis. These findings identify an Aurora B-dependent mechanism that implicates Ataxin-10 in cytokinesis.
doi:10.1038/srep08360
PMCID: PMC4322367  PMID: 25666058
5.  Denatonium inhibits growth and induces apoptosis of airway epithelial cells through mitochondrial signaling pathways 
Respiratory Research  2015;16(1):13.
Background
Denatonium, a widely used bitter agonist, activates bitter taste receptors on many cell types and plays important roles in chemical release, ciliary beating and smooth muscle relaxation through intracellular Ca2+-dependent pathways. However, the effects of denatonium on the proliferation of airway epithelial cells and on the integrity of cellular components such as mitochondria have not been studied. In this study, we hypothesize that denatonium might induce airway epithelial cell injury by damaging mitochondria.
Methods
Bright-field microscopy, cell counting kit-8 (CCK-8) assay and flow cytometry analysis were used to examine cellular morphology, proliferation and cell cycle, respectively. Transmission electron microscopy (TEM) was used to examine mitochondrial integrity. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and protein expression, respectively.
Results
For airway epithelial cells, we observed that denatonium significantly effects cellular morphology, decreases cell proliferation and reduces the number of cells in S phase in a dose-dependent manner. TEM analysis demonstrated that denatonium causes large amplitude swelling of mitochondria, which was confirmed by the loss of mitochondrial membrane potential, the down-regulation of Bcl-2 protein and the subsequent enhancement of the mitochondrial release of cytochrome c and Smac/DIABLO after denatonium treatment.
Conclusions
In this study, we demonstrated for the first time that denatonium damages mitochondria and thus induces apoptosis in airway epithelial cells.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-015-0183-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12931-015-0183-9
PMCID: PMC4326484  PMID: 25652218
Denatonium; Bitter taste receptors; Epithelium injury; Mitochondria; Cytochrome c
6.  Covalent Modification of a Cysteine Residue in the XPB Subunit of the General Transcription Factor TFIIH Through Single Epoxide Cleavage of the Transcription Inhibitor Triptolide** 
Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these results deepen our understanding of the interaction between triptolide and XPB and have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs.
doi:10.1002/anie.201408817
PMCID: PMC4314353  PMID: 25504624
inhibitors; medicinal chemistry; natural products; target validation; transcription factors
7.  Dysbiosis gut microbiota associated with inflammation and impaired mucosal immune function in intestine of humans with non-alcoholic fatty liver disease 
Scientific Reports  2015;5:8096.
Non-alcoholic fatty liver disease (NAFLD) has recently been considered to be under the influence of the gut microbiota, which might exert toxic effects on the human host after intestinal absorption and delivery to the liver via the portal vein. In this study, the composition of the gut microbiota in NAFLD patients and healthy subjects was determined via 16S ribosomal RNA Illumina next-generation sequencing. Among those taxa displaying greater than 0.1% average abundance in all samples, five genera, including Alistipes and Prevotella, were significantly more abundant in the gut microbiota of healthy subjects compared to NAFLD patients. Alternatively, Escherichia, Anaerobacter, Lactobacillus and Streptococcus were increased in the gut microbiota of NAFLD patients compared to healthy subjects. In addition, decreased numbers of CD4+ and CD8+ T lymphocytes and increased levels of TNF-α, IL-6 and IFN-γ were detected in the NAFLD group compared to the healthy group. Furthermore, irregularly arranged microvilli and widened tight junctions were observed in the gut mucosa of the NAFLD patients via transmission electron microscopy. We postulate that aside from dysbiosis of the gut microbiota, gut microbiota-mediated inflammation of the intestinal mucosa and the related impairment in mucosal immune function play an important role in the pathogenesis of NAFLD.
doi:10.1038/srep08096
PMCID: PMC4314632  PMID: 25644696
8.  Field-based evidence for consistent responses of bacterial communities to copper contamination in two contrasting agricultural soils 
Copper contamination on China's arable land could pose severe economic, ecological and healthy consequences in the coming decades. As the drivers in maintaining ecosystem functioning, the responses of soil microorganisms to long-term copper contamination in different soil ecosystems are still debated. This study investigated the impacts of copper gradients on soil bacterial communities in two agricultural fields with contrasting soil properties. Our results revealed consistent reduction in soil microbial biomass carbon (SMBC) with increasing copper levels in both soils, coupled by significant declines in bacterial abundance in most cases. Despite of contrasting bacterial community structures between the two soils, the bacterial diversity in the copper-contaminated soils showed considerably decreasing patterns when copper levels elevated. High-throughput sequencing revealed copper selection for major bacterial guilds, in particular, Actinobacteria showed tolerance, while Acidobacteria and Chloroflexi were highly sensitive to copper. The thresholds that bacterial communities changed sharply were 800 and 200 added copper mg kg−1 in the fluvo-aquic soil and red soil, respectively, which were similar to the toxicity thresholds (EC50 values) characterized by SMBC. Structural equation model (SEM) analysis ascertained that the shifts of bacterial community composition and diversity were closely related with the changes of SMBC in both soils. Our results provide field-based evidence that copper contamination exhibits consistently negative impacts on soil bacterial communities, and the shifts of bacterial communities could have largely determined the variations of the microbial biomass.
doi:10.3389/fmicb.2015.00031
PMCID: PMC4313605
copper contamination; soil bacterial community; diversity; abundance; community composition; soil microbial biomass carbon; field experiment
9.  Spinal NF-κB and Chemokine Ligand 5 Expression during Spinal Glial Cell Activation in a Neuropathic Pain Model 
PLoS ONE  2015;10(1):e0115120.
Background
The NF-κB pathway and chemokine (C-C motif) ligand 5 (CCL5) are involved in pain modulation; however, the precise mechanisms of their interactions in chronic neuropathic pain have yet to be established.
Methods
The present study examined the roles of spinal NF-κB and CCL5 in a neuropathic pain model after chronic constriction injury (CCI) surgery. CCI-induced pain facilitation was evaluated using the Plantar and von Frey tests. The changes in NF-κB and CCL5 expression were analyzed by immunohistochemistry and Western blot analyses.
Results
Spinal NF-κB and CCL5 expression increased after CCI surgery. Repeated intrathecal infusions of pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) decreased CCL5 expression, inhibited the activation of microglia and astrocytes, and attenuated CCI-induced allodynia and hyperalgesia. Intrathecal injection of a CCL5-neutralizing antibody attenuated CCI-induced pain facilitation and also suppressed spinal glial cell activation after CCI surgery. However, the CCL5-neutralizing antibody did not affect NF-κB expression. Furthermore, selective glial inhibitors, minocycline and fluorocitrate, attenuated the hyperalgesia induced by intrathecal CCL5.
Conclusions
The inhibition of spinal CCL5 expression may provide a new method to prevent and treat nerve injury-induced neuropathic pain.
doi:10.1371/journal.pone.0115120
PMCID: PMC4312098  PMID: 25635831
10.  Meta-Analysis of Anti-Muscarinic Receptor Type 3 Antibodies for the Diagnosis of Sjögren Syndrome 
PLoS ONE  2015;10(1):e0116744.
Purpose
To conduct a meta-analysis to evaluate the diagnostic value of anti-muscarinic receptor type 3 (M3R) antibodies in Sjögren syndrome (SS).
Methods
Two databases, PUBMED and the Cochrane Library, were systematically searched. Approximately 2,000 participants from several studies were included in this research. STATA 11.2 software and Meta-DiSc 1.4 was used to conduct the meta-analysis.
Results
Eleven studies were included in the meta-analysis. The pooled DOR was 13.00 (95% CI, 6.00–26.00). The sensitivity was 0.43 (95% CI, 0.28–0.58) and the specificity was 0.95 (95%CI, 0.91–0.97). The LR+ and LR- were 7.90 (95% CI, 4.70–13.40), 0.61 (95% CI, 0.46–0.79), respectively. The AUC was 0.89 (95% CI, 0.86–0.92).
Conclusion
The anti-M3R antibody had high specificity but relatively low sensitivity for the diagnosis of SS.
doi:10.1371/journal.pone.0116744
PMCID: PMC4309563  PMID: 25629973
11.  Exosomes: Novel Biomarkers for Clinical Diagnosis 
The Scientific World Journal  2015;2015:657086.
Exosomes are 30–120 nm endocytic membrane-derived vesicles that participate in cell-to-cell communication and protein and RNA delivery. Exosomes harbor a variety of proteins, nucleic acids, and lipids and are present in many and perhaps all bodily fluids. A significant body of literature has demonstrated that molecular constituents of exosomes, especially exosomal proteins and microRNAs (miRNAs), hold great promise as novel biomarkers for clinical diagnosis. In this minireview, we summarize recent advances in the research of exosomal biomarkers and their potential application in clinical diagnostics. We also provide a brief overview of the formation, function, and isolation of exosomes.
doi:10.1155/2015/657086
PMCID: PMC4322857
12.  Effects of whole cigarette smoke on human beta defensins expression and secretion by oral mucosal epithelial cells 
Tobacco Induced Diseases  2015;13(1):3.
Background
Cigarette smoke a recognized risk factor for many systemic diseases and also oral diseases. Human beta defensins (HBDs), a group of important antimicrobial peptides expressed by the epithelium, are crucial for local defense and tissue homeostasis of oral cavity. The aim of this study was to evaluate potential effects of whole cigarette smoke (WCS) exposure on the expression and secretion of HBDs by oral mucosal epithelial cells.
Methods
Immortalized human oral mucosal epithelial (Leuk-1) cells were exposed to WCS for various time periods. HBD-1, -2 and -3 expression and subcellular localization were detected by real time qPCR, immunofluorescence assay and confocal microscopy. According to the relative fluorescent intensity, the expression levels of HBD-1, -2 and -3 were evaluated by digital image analysis system. The alteration of HBD-1, -2 and -3 secretion levels was measured by the Enzyme-Linked Immunosorbent Assay.
Results
WCS exposure remarkably attenuated HBD-1 expression and secretion while clearly enhanced HBD-2, -3 expression levels and HBD-2 secretion by Leuk-l cells. It appeared that there was no significant effect of WCS exposure on HBD-3 secretion.
Conclusions
WCS exposure could modulate expression and secretion of HBDs by oral mucosal epithelial cells, establishing a link between cigarette smoke and abnormal levels of antimicrobial peptides. The present results may give a new perspective to investigate smoking-related local defense suppression and oral disease occurrence.
doi:10.1186/s12971-015-0029-8
PMCID: PMC4310021  PMID: 25635179
Whole cigarette smoke; Human β defensin; Oral mucosa
13.  Tumor-targeting novel manganese complex induces ROS-mediated apoptotic and autophagic cancer cell death 
In this study, the antitumor activity of the novel manganese (II) compound, Adpa-Mn {[(Adpa)Mn(Cl)(H2O)] (Adpa=bis(2-pyridylmethyl)amino-2-propionic acid)}, and its possible mechanisms of action were investigated. In vitro, the growth inhibitory effects of Adpa-Mn (with IC50 values lower than 15 μM) on tumor cell lines were examined by MTT assay. We found that this compound was more selective against cancer cells than the popular chemotherapeutic reagent, cisplatin. We then found that Adpa-Mn achieved its selectivity against cancer cells through the transferrin (Tf)-transferrin receptor (TfR) system, which is highly expressed in tumor cells. Furthermore, Adpa-Mn induced both apoptosis and autophagy, as indicated by chromatin condensation, the activation of poly(ADP-ribose) polymerase (PARP), Annexin V/prop-idium iodide staining, an enhanced fluorescence intensity of monodansylcadaverine (MDC), as well as the elevated expression of the autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3). In addition, Adpa-Mn induced the generation of intracellular reactive oxygen species (ROS) and its anticancer effects were significantly reduced following pre-treatment with the antioxidant, N-acetyl cysteine, indicating that ROS triggered cell death. In vivo, the induction of apoptosis and autophagy in tumor tissue was confirmed following treatment with Adpa-Mn, which contributed to its significant antitumor activity against hepatocellular carcinoma (Hep-A cell) xenografts at 10 mg/kg. Taken together, these data suggest the possible use of Adpa-Mn as a novel anticancer drug.
doi:10.3892/ijmm.2015.2073
PMCID: PMC4314420  PMID: 25604962
manganese complex; anticancer; autophagy; apoptosis; reactive oxygen species
14.  ADAMTS-7 Exhibits Elevated Expression in Cartilage of Osteonecrosis of Femoral Head and Has a Positive Correlation with TNF-α and NF-κB P65 
Mediators of Inflammation  2015;2015:196702.
ADAMTS-7 has been reported to exaggerate cartilage degeneration and to be associated with TNF-α and NF-κB signaling pathway. In this study we compared the expression of ADAMTS-7, TNF-α, and Phospho-NF-κB in patients with femoral neck fracture (FNF) and osteonecrosis of femoral head (ONFH) at different stages. We found that expression of ADAMTS-7, TNF-α, and Phospho-NF-κB was significantly upregulated in ONFH patients' articular cartilage and related to the pathogenesis of ONFH. Thus we conclude that ADAMTS-7 level appears to be positively associated with expression of TNF-α and Phospho-NF-κB P65 in cartilage, which may imply its association with cartilage destruction of ONFH.
doi:10.1155/2015/196702
PMCID: PMC4310498  PMID: 25653475
15.  Successful management of acute myeloid leukemia transformed from chronic myelomonocytic leukemia in the elderly by a combination regimen of decitabine and cytarabine, aclarubicin and granulocyte colony-stimulating factor: A case report 
Oncology Letters  2015;9(3):1217-1220.
Despite advances in the treatment of acute myeloid leukemia (AML) in recent years, the outcome of elderly AML patients with antecedent hematological disorders remains unsatisfactory. The present study describes a case of complete remission in an elderly patient with AML transformed from chronic myelomonocytic leukemia (CMML) and the treatment of the case with decitabine in combination with cytarabine, aclarubicin and granulocyte colony-stimulating factor (CAG). A 70-year-old male was admitted with fever, pruritus and weakness that had been apparent for two weeks, and a two-year history of monocytosis (22.5–27.0%). Further examinations revealed a hemoglobin level of 106 g/l, a white blood cell count of 39.52×109/l, a platelet count of 81×109/l, Y chromosome loss and uniparental disomy on chromosomes 4q, 2q and 19p. The patient was diagnosed with AML transformed from CMML, with cytogenetic anomalies. A combination regimen of decitabine and CAG was administered. Subsequent to one cycle, the patient achieved complete remission. The patient was then followed up with three courses of the same regimen and achieved clinical remission, with no evidence of AML relapse. The present study suggests that a combination of low-dose decitabine and CAG may offer a novel and potentially effective treatment regimen for elderly AML patients.
doi:10.3892/ol.2015.2870
PMCID: PMC4315126  PMID: 25663885
acute myeloid leukemia; chronic myeloid leukemia; decitabine; cytarabine; aclarubicin and granulocyte colony-stimulating factor; single nucleotide polymorphism
16.  Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate ON 01910.Na, a mitotic progression modulator, in human plasma 
A reverse-phase high performance liquid chromatographic method with tandem mass spectrometry (LC-MS/MS) was developed and validated for the quantitation of ON 01910.Na, a novel synthetic benzyl styryl sulfone, in human plasma. The assay involved a simple sample preparation with acetonitrile protein precipitation. ON 01910.Na and the internal standard temazepam were separated on a Waters X-Terra™ MS C18 column with mobile phase of acetonitrile containing 0.1% formic acid /10 mM ammonium acetate (55:45, v/v) using isocratic flow at 0.2 mL/min for 5 minutes. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Two calibration curves were generated over the range of 10–2000 ng/mL and 100–20000 ng/mL. The lower limit of quantitation (LLOQ) was 10 ng/mL for ON 01910.Na in human plasma. The accuracy and within- and between-day precisions were within the acceptance criteria for bioanalytical assays. ON 01910.Na was found stable in plasma at −70°C for at least 1 year. The method was successfully applied to characterize the plasma concentration-time profiles of ON 01910.Na in the cancer patients in the phase I study.
doi:10.1016/j.jchromb.2007.05.047
PMCID: PMC4286291  PMID: 17588831
ON 01910.Na; high performance liquid chromatography (HPLC); mass spectrometry (MS); LC-MS/MS; pharmacokinetics
17.  Comparison of Postural Responses to Galvanic Vestibular Stimulation between Pilots and the General Populace 
BioMed Research International  2015;2015:567690.
Galvanic vestibular stimulation (GVS) can be used to study the body's response to vestibular stimuli. This study aimed to investigate whether postural responses to GVS were different between pilots and the general populace. Bilateral bipolar GVS was applied with a constant-current profile to 12 pilots and 12 control subjects via two electrodes placed over the mastoid processes. Both GVS threshold and the center of pressure's trajectory (COP's trajectory) were measured. Position variability of COP during spontaneous body sway and peak displacement of COP during GVS-induced body sway were calculated in the medial-lateral direction. Spontaneous body sway was slight for all subjects, and there was no significant difference in the value of COP position variability between the pilots and controls. Both the GVS threshold and magnitude of GVS-induced body deviation were similar for different GVS polarities. GVS thresholds were similar between the two groups, but the magnitude of GVS-induced body deviation in the controls was significantly larger than that in the pilots. The pilots showed less GVS-induced body deviation, meaning that pilots may have a stronger ability to suppress vestibular illusions.
doi:10.1155/2015/567690
PMCID: PMC4302968  PMID: 25632395
18.  Expression of a long variant of CRACR2A that belongs to the Rab GTPase protein family in endothelial cells 
Highlights
•Knockdown of CRACR2A lacks effect on CRAC channels in endothelial cells.•Knockdown of CRACR2A depletes a protein twice the mass of CRACR2A.•A long variant of CRACR2A, CRACR2A-L, occurs in endothelial cells.•CRACR2A-L is a previously unrecognised EF-hand-containing Rab GTPase.•CRACR2A-L has a positive role in endothelial tube formation.
CRACR2A protein is described in T cells as an EF-hand-containing modulator of calcium-release-activated calcium (CRAC) channels. Here we sought relevance to calcium entry of endothelial cells. Unexpectedly, short interfering RNA designed to deplete CRACR2A had no effect on CRAC channels in endothelial cells but reduced the abundance of a protein with about twice the mass of CRACR2A. Reference to gene sequence data indicated the potential for a variant transcript encoding a C-terminal Rab GTPase extension of CRACR2A. Full-length cloning demonstrated expression of the long variant in endothelial cells. It was designated CRACR2A-L. Sequence analysis suggested it to be a previously unrecognised member of the Rab GTPase family. It made a positive contribution to endothelial tube formation. The data suggest that endothelial cells contain a long variant of CRACR2A which is an EF-hand-containing Rab protein that lacks impact on CRAC channels.
doi:10.1016/j.bbrc.2014.11.095
PMCID: PMC4300414  PMID: 25475730
CRAC channel, calcium-release-activated calcium channel; CRACR2A, CRAC channel regulator 2A; CRACR2A-L, CRACR2A long; HUVEC, human umbilical vein endothelial cell; Calcium channel; Store-operated calcium entry; G protein; Endothelial cell; Angiogenesis
19.  Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents 
Bioorganic & medicinal chemistry  2013;22(1):116-125.
A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alternations to the C19-C22 Z, E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells.
doi:10.1016/j.bmc.2013.11.046
PMCID: PMC3958936  PMID: 24359706
Pateamine A; DMDAPatA; Stille coupling; Translation initiation; eIF4A
20.  Characterization of the Nucleocytoplasmic Shuttle of the Matrix Protein of Influenza B Virus 
Journal of Virology  2014;88(13):7464-7473.
ABSTRACT
Influenza B virus is an enveloped negative-strand RNA virus that contributes considerably to annual influenza illnesses in human. The matrix protein of influenza B virus (BM1) acts as a cytoplasmic-nuclear shuttling protein during the early and late stages of infection. The mechanism of this intracellular transport of BM1 was revealed through the identification of two leucine-rich CRM1-dependent nuclear export signals (NESs) (3 to 14 amino acids [aa] and 124 to 133 aa), one bipartite nuclear localization signal (NLS) (76 to 94 aa), and two phosphorylation sites (80T and 84S) in BM1. The biological function of the NLS and NES regions were determined through the observation of the intracellular distribution of enhanced green fluorescent protein (EGFP)-tagged signal peptides, and wild-type, NES-mutant, and NLS-mutant EGFP-BM1. Furthermore, the NLS phosphorylation sites 80T and 84S, were found to be required for the nuclear accumulation of EGFP-NLS and for the efficient binding of EGFP-BM1 to human importin-α1. Moreover, all of these regions/sites were required for the generation of viable influenza B virus in a 12-plasmid virus rescue system.
IMPORTANCE This study expands our understanding of the life cycle of influenza B virus by defining the dynamic mechanism of the nucleocytoplasmic shuttle of BM1 and could provide a scientific basis for the development of antiviral medication.
doi:10.1128/JVI.00794-14
PMCID: PMC4054458  PMID: 24741102
21.  Characteristics of Nucleocytoplasmic Transport of H1N1 Influenza A Virus Nuclear Export Protein 
Journal of Virology  2014;88(13):7455-7463.
ABSTRACT
The influenza A virus nuclear export protein (NEP) plays crucial roles in the nuclear export of the viral ribonucleoprotein complex through the chromosome region maintenance 1 (CRM1)-mediated cellular protein transport system. However, the detailed mechanism of NEP nucleocytoplasmic trafficking remains incompletely understood. Here, we investigated the subcellular localization of NEP from two strains of H1N1 influenza A virus and found that 2009 swine-origin H1N1 influenza A virus A/California/04/2009 (CA04) NEP displayed a distinct cellular distribution pattern, forming unique nuclear aggregates, compared to A/WSN/33 (H1N1) (WSN) NEP. Characterization of the nucleocytoplasmic transport pathways of these two NEPs showed that they both enter the nucleus by passive diffusion but are exported through the nuclear export receptor CRM1-mediated pathway with different efficiencies. The two identified nuclear export signals (NESs) on the two NEPs functioned similarly despite differences in their amino acid sequences. Using a two-hybrid assay, we confirmed that the CA04 NEP interacts less efficiently with CRM1 and that a threonine residue at position 48 is responsible for the nuclear aggregation. The present study revealed the dissimilarity in subcellular NEP transport processes between the 2009 pandemic (H1N1) influenza A virus CA04 and the laboratory-adapted H1N1 virus WSN and uncovered the mechanism responsible for this difference.
IMPORTANCE Because the efficiency of the nucleocytoplasmic transport of viral components is often correlated with the viral RNA polymerase activity, propagation, and host range of influenza viruses, the present study investigated the subcellular localization of NEP from two strains of H1N1 influenza virus. We found that the NEPs of both A/California/04/2009 (H1N1) (CA04) and A/WSN/33 (H1N1) (WSN) enter the nucleus by passive diffusion but are exported with different efficiencies, which were caused by weaker binding activity between the CA04 NEP and CRM1. The results of the present study revealed characteristics of the nuclear import and export pathways of NEP and the mechanism responsible for the differences in the cellular distribution of NEP between two H1N1 strains.
doi:10.1128/JVI.00257-14
PMCID: PMC4054460  PMID: 24741105
22.  Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target 
Cancer Biology & Therapy  2013;15(1):128-134.
Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.
doi:10.4161/cbt.27146
PMCID: PMC3938515  PMID: 24253382
gastric cancer; lung cancer; PPME1 amplification; PP2A; shRNA-knockdown
23.  Efficacy and safety of individually tailored antiplatelet therapy in patients with acute coronary syndrome after coronary stenting: a single center, randomized, feasibility study 
Background
Low responsiveness to clopidogrel (LRC) is associated with increased risk of ischemic events. This study was aimed to explore the feasibility of tailored antiplatelet therapy according to the responsiveness to clopidogrel.
Methods
A total of 305 clopidogrel naïve patients with acute coronary syndromes (ACS) undergoing coronary stenting were randomly assigned to receive standard (n = 151) or tailored (n = 154) antiplatelet therapy. The ADP-induced platelet aggregation tests by light transmission aggregometry were performed to identify LRC patients assigned to the tailored group. The standard antiplatelet regimen was dual antiplatelet therapy with aspirin and clopidogrel. The tailored antiplatelet therapy was standard regimen for non-LRC patients and an additional 6-month cilostazol treatment for LRC patients. The primary efficacy outcome was the composite of cardiovascular death, myocardial infarction or stroke at one year.
Results
LCR was present in 26.6% (41/154) of patients in the tailored group. The percentage platelet aggregation for LCR patients was significantly decreased at three days after adjunctive cilostazol treatment (77.5% ± 12.1% vs. 64.5% ± 12.1%, P < 0.001). At one year follow-up, a non-significant 37% relative risk reduction of primary events were observed in the tailored group as compared to the standard group (5.8% vs. 9.3%, P = 0.257). There were no differences in the rates of stent thrombosis and hemorrhagic events between the two groups.
Conclusions
Tailored antiplatelet therapy for ACS patients after coronary stenting according to responsiveness to clopidogrel is feasible. However, its efficacy and safety need further confirmation by clinical trials with larger sample sizes.
doi:10.11909/j.issn.1671-5411.2015.01.003
PMCID: PMC4308455
Acute coronary syndrome; Antiplatelet therapy; Clopidogrel; Coronary stenting
24.  Non-Enzymatic Depurination of Nucleic Acids: Factors and Mechanisms 
PLoS ONE  2014;9(12):e115950.
Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC). Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius’ law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA) was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination.
doi:10.1371/journal.pone.0115950
PMCID: PMC4278771  PMID: 25546310
25.  A Comparison between the Sixth and Seventh Editions of the UICC/AJCC Staging System for Nasopharyngeal Carcinoma in a Chinese Cohort 
PLoS ONE  2014;9(12):e116261.
Background
The International Union Against Cancer/American Joint Committee on Cancer (UICC/AJCC) TNM staging system of nasopharyngeal carcinoma (NPC) is the most important system for survival prediction. The TNM 7th edition UICC/AJCC TNM staging system for NPC was adopted in January 2009, and is now internationally recommended. In comparison with the TNM 6th edition, there were several revisions in the new edition staging system. This study aims to evaluate the prognostic value of the TNM 7th edition for NPC patients in comparison with the TNM 6th edition.
Method
Clinical data of 2,629 NPC patients from the Sun Yat-sen University Cancer Center between January 2006 and December 2010 were retrospectively collected and all the patients were restaged according to the criteria of the TNM 6th edition and TNM 7th edition UICC/AJCC staging manual. Univariate and multivariate COX proportional hazards analyses were applied to evaluate the prognostic values between adjacent stage categories of the TNM 6th edition and TNM 7th edition.
Results
In comparison with the TNM 6th edition, a significant alteration of the distribution of N categories was observed when the TNM 7th edition was applied (χ2 = 20.589, P<0.001), with 119 (119/670, 17.8%) patients up-staging from N0 to N1. With regard to T and overall stage, 37 (37/561, 6.6%) patients were down-staged from T2a with the TNM 6th edition to T1 with the TNM 7th edition, and finally two patients were up-staged to overall stage II (2/118, 1.7%). Moreover, the survival curves were significantly segregated (P<0.05) between T1 and T2 as well as N1 and N2 with the TNM 7th edition.
Conclusions
The TNM 7th edition led to a significant alteration in the distribution of N categories and it is superior to the TNM 6th edition in predicting the frequency of overall survival and distant metastasis-free survival.
doi:10.1371/journal.pone.0116261
PMCID: PMC4275293  PMID: 25536307

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