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1.  Reactivation of the homeotic tumor suppressor gene CDX2 by 5-aza-2′-deoxycytidine-induced demethylation inhibits cell proliferation and induces caspase-independent apoptosis in gastric cancer cells 
The DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is widely used as an anticancer drug for the treatment of leukemia and solid tumors. Gastric cancer (GC) patients who were positive for caudal type homeobox transcription factor 2 (CDX2) expression showed a higher survival rate compared with those who were CDX2 negative, which suggests that CDX2 performs a tumor suppressor role. However, the molecular mechanisms leading to the inactivation of CDX2 remain unclear. In the present study we demonstrated that the expression levels of CDX2 and DNA methyltransferase enzyme 1 (DNMT1) mRNA were significantly higher in GC compared with distal non-cancerous tissue. The expression of CDX2 mRNA was significantly correlated with Lauren classification, TNM stage and lymph node metastasis. DNMT1 mRNA expression was significantly correlated with TNM stage, pathological differentiation and lymph node metastasis. The expression of CDX2 mRNA was inversely correlated with that of DNMT1 mRNA in GC. Hypermethylation of the CDX2 gene promoter region, extremely low expression levels of CDX2 mRNA and no expression of CDX2 protein were the characteristics observed in MKN-45 and SGC-7901 GC cell lines. Following the treatment of MKN-45 cells with 5-aza-CdR, the hypermethylated CDX2 gene promoter region was demethylated and expression of CDX2 was upregulated, while DNMT1 expression was downregulated. Furthermore, a concentration- and time-dependent growth inhibition as well as increased apoptosis were observed. Caspase-3, −8 and −9 activities increased in a concentration-dependent manner following exposure to different concentrations of 5-aza-CdR. Therefore, our data show that the overexpression of DNMT1 and methylation of the CDX2 gene promoter region is likely to be responsible for CDX2 silencing in GC. 5-Aza-CdR may effectively induce re-expression of the CDX2 gene, inhibit cell proliferation and enhance the caspase-independent apoptosis of MKN-45 cells in vitro.
PMCID: PMC3570199  PMID: 23408490
gastric neoplasms; caudal type homeobox transcription factor 2; DNA methylation; DNA methyltransferase enzyme 1; proliferation; apoptosis
2.  Using immunoproteomics to identify alpha-enolase as an autoantigen in liver fibrosis 
Journal of proteome research  2013;12(4):1789-1796.
Liver fibrosis results from extracellular matrix accumulation during the wound healing process when the liver is insulted with chronic viral infection, inflammation, or alcoholic diseases. The current diagnosis of liver fibrosis is mainly dependent on biopsy, which is an invasive approach. Identification of serological biomarkers has been considered as the most promising way for early detection of the disease. Although several biomarkers in liver fibrosis have been identified, the problem is that these markers can be also detected in fibrogenesis which occurred in other organs. In this study, we have identified and characterized some cellular proteins which can be recognized by autoantibodies in the sera from patients with pre-cirrhotic stage of liver fibrosis. Among 180 sera from patients with liver fibrosis, 14.4% (26/180) of sera contained autoantibody against a protein migrating around 47-kDa on SDS-PAGE gel. Indirect immunofluorescence assay using purified autoantibody against the 47-kDa protein showed that this protein mainly localized in the cytoplasm. Using immunoproteomic approach, the 47-kDa protein was identified as alpha-enolase. In further study, the frequency of anti-alpha-enolase antibody in sera from patients with pre-cirrhotic stage of liver fibrosis (21.6%, 27/125) was significantly higher than that in sera from patients with cirrhosis (9.1%, 5/55) and liver cancer (14.3%, 12/84), as well as in sera from healthy individuals (4.1%, 3/74). Therefore, alpha-enolase is an autoantigen that elicits autoimmune response in liver fibrosis and can be a potential prognostic factor for liver fibrosis diagnosis.
PMCID: PMC3743961  PMID: 23458688
liver fibrosis; autoantigen; autoimmune response; immunoproteomics; alpha-enolase
3.  Establishing and Applying a Schistosomiasis Early Warning Index (SEWI) in the Lower Yangtze River Region of Jiangsu Province, China 
PLoS ONE  2014;9(4):e94012.
China has made remarkable progress in schistosomiasis control over the past decades. Transmission control has replaced morbidity control as the country moves towards the goal of elimination and the current challenge is to find a sensitive measure capable of gauging transmission risk in low-prevalence areas. The study aims to develop a Schistosomiasis Early Warning Index (SEWI) and demonstrate its use in Jiangsu Province along the lower Yangtze River.
Methodology/Principal Findings
The Delphi approach, a structured communication technique, was used to develop the SEWI. Two rounds of interviews with 30 public health experts specialized in schistosomiasis control were conducted using 40 indicators that reflected different aspects of schistosomiasis transmission and control. The necessity, feasibility, and sensitivity of each indicator were assessed and the weight value of each indicator determined based on these experts' judgment. The system included 3 first-order indicators, 7 second-order indicators, and 30 third-order indicators. The 3 first-order indicators were endemic status, control measures, social and environmental factors, with the weight values 0.366, 0.343 and 0.291, respectively. For the 7 second-order indicators, the highest weight value was for control measures for snails (0.175) and the lowest for transmission route (0.110). We estimated and mapped the SEWI for endemic areas at the county scale in Jiangsu Province finding that the majority of the endemic areas were characterized as medium transmission risk (SEWI risk values between 0.3 and 0.6), while areas where transmission interruption had been officially declared showed SEWI values <0.30. A few isolated areas (e.g. endemic islands in the Yangtze River) produced SEWI values >0.60. These estimates are largely in agreement with the endemicity levels based on recent epidemiological surveys.
The SEWI should be useful for estimation of schistosomiasis transmission surveillance, particularly with reference to the elimination of the disease in China.
PMCID: PMC3976384  PMID: 24705352
4.  VEGF controls lung Th2 inflammation via the miR-1–Mpl (myeloproliferative leukemia virus oncogene)–P-selectin axis 
The Journal of Experimental Medicine  2013;210(10):1993-2010.
VEGF dampens the expression of microRNA-1, which drives inflammation in part via increasing the expression of Mpl.
Asthma, the prototypic Th2-mediated inflammatory disorder of the lung, is an emergent disease worldwide. Vascular endothelial growth factor (VEGF) is a critical regulator of pulmonary Th2 inflammation, but the underlying mechanism and the roles of microRNAs (miRNAs) in this process have not been defined. Here we show that lung-specific overexpression of VEGF decreases miR-1 expression in the lung, most prominently in the endothelium, and a similar down-regulation occurs in lung endothelium in Th2 inflammation models. Intranasal delivery of miR-1 inhibited inflammatory responses to ovalbumin, house dust mite, and IL-13 overexpression. Blocking VEGF inhibited Th2-mediated lung inflammation, and this was restored by antagonizing miR-1. Using mRNA arrays, Argonaute pull-down assays, luciferase expression assays, and mutational analysis, we identified Mpl as a direct target of miR-1 and showed that VEGF controls the expression of endothelial Mpl during Th2 inflammation via the regulation of miR-1. In vivo knockdown of Mpl inhibited Th2 inflammation and indirectly inhibited the expression of P-selectin in lung endothelium. These experiments define a novel VEGF–miR-1–Mpl–P-selectin effector pathway in lung Th2 inflammation and herald the utility of miR-1 and Mpl as potential therapeutic targets for asthma.
PMCID: PMC3782056  PMID: 24043765
5.  Recruitment of Cbl-b to B Cell Antigen Receptor Couples Antigen Recognition to Toll-Like Receptor 9 Activation in Late Endosomes 
PLoS ONE  2014;9(3):e89792.
Casitas B-lineage lymphoma-b (Cbl-b) is a ubiquitin ligase (E3) that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR) and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA) domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9) into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.
PMCID: PMC3961229  PMID: 24651487
6.  Cisplatin improves antitumor activity of weekly nab-paclitaxel in patients with metastatic breast cancer 
Although nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is approved to be given every 3 weeks, weekly use of this drug is becoming a new standard of care in patients with metastatic breast cancer (MBC). This prospective Phase II study was conducted to improve the efficacy of weekly nab-paclitaxel with cisplatin in MBC patients. Seventy-three women with recurrent or MBC were eligible for participation. Nab-paclitaxel was administered weekly at a dose of 125 mg/m2 on day 1, day 8, and day 15, followed by cisplatin 75 mg/m2 on day 1, repeated every 28 days with a maximum of 6 cycles. The primary objective was investigator-assessed overall response rate (ORR). A high ORR of 67.1% was obtained, with rates of 80.6% for the first-line patients and 80% for patients not pretreated with taxanes. Among those who had objective responses, a large percentage of patients (83.7%) showed quickly remarkable tumor shrinkage during the first two cycles. The median progression-free and overall survival times were 9.8 and 26.9 months, respectively. For the patients receiving first-, second-, and third-line therapy or beyond, median progression-free survival was 11.7, 7.7, and 7.6 months, respectively (P=0.005). Molecular subtype was not significantly associated with ORR or disease progression. Grade 4 neutropenia occurred in 46 patients (63.0%), with febrile neutropenia found in 9 patients (12.3%). Grade 3 peripheral neuropathy was an accumulated dose-limiting toxicity occurring in 19 patients (26.0%). Efficacy of weekly nab-paclitaxel can be improved by adding cisplatin. The doublet is highly effective, with quick response, manageable toxicity, and possible equivalence across molecular subtypes in MBC patients.
PMCID: PMC3964033  PMID: 24672237
metastatic breast cancer; nanoparticle albumin-bound paclitaxel; cisplatin; taxane-pretreated
7.  Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells 
PLoS ONE  2014;9(3):e91557.
Hematopoiesis is a complex process regulated by sets of transcription factors in a stage-specific and context-dependent manner. THAP11 is a transcription factor involved in cell growth, ES cell pluripotency, and embryogenesis. Here we showed that THAP11 was down-regulated during erythroid differentiation but up-regulated during megakaryocytic differentiation of cord blood CD34+ cells. Overexpression of THAP11 in K562 cells inhibited the erythroid differentiation induced by hemin with decreased numbers of benzidine-positive cells and decreased mRNA levels of α-globin (HBA) and glycophorin A (GPA), and knockdown of THAP11 enhanced the erythroid differentiation. Conversely, THAP11 overexpression accelerated the megakaryocytic differentiation induced by phorbol myristate acetate (PMA) with increased percentage of CD41+ cells, increased numbers of 4N cells, and elevated CD61 mRNA levels, and THAP11 knockdown attenuated the megakaryocytic differentiation. The expression levels of transcription factors such as c-Myc, c-Myb, GATA-2, and Fli1 were changed by THAP11 overexpression. In this way, our results suggested that THAP11 reversibly regulated erythroid and megakaryocytic differentiation.
PMCID: PMC3956667  PMID: 24637716
8.  Streptococcus iniae SF1: Complete Genome Sequence, Proteomic Profile, and Immunoprotective Antigens 
PLoS ONE  2014;9(3):e91324.
Streptococcus iniae is a Gram-positive bacterium that is reckoned one of the most severe aquaculture pathogens. It has a broad host range among farmed marine and freshwater fish and can also cause zoonotic infection in humans. Here we report for the first time the complete genome sequence as well as the host factor-induced proteomic profile of a pathogenic S. iniae strain, SF1, a serotype I isolate from diseased fish. SF1 possesses a single chromosome of 2,149,844 base pairs, which contains 2,125 predicted protein coding sequences (CDS), 12 rRNA genes, and 45 tRNA genes. Among the protein-encoding CDS are genes involved in resource acquisition and utilization, signal sensing and transduction, carbohydrate metabolism, and defense against host immune response. Potential virulence genes include those encoding adhesins, autolysins, toxins, exoenzymes, and proteases. In addition, two putative prophages and a CRISPR-Cas system were found in the genome, the latter containing a CRISPR locus and four cas genes. Proteomic analysis detected 21 secreted proteins whose expressions were induced by host serum. Five of the serum-responsive proteins were subjected to immunoprotective analysis, which revealed that two of the proteins were highly protective against lethal S. iniae challenge when used as purified recombinant subunit vaccines. Taken together, these results provide an important molecular basis for future study of S. iniae in various aspects, in particular those related to pathogenesis and disease control.
PMCID: PMC3951389  PMID: 24621602
9.  ABCB1 1199G>A Genetic Polymorphism (Rs2229109) Influences the Intracellular Accumulation of Tacrolimus in HEK293 and K562 Recombinant Cell Lines 
PLoS ONE  2014;9(3):e91555.
ATP-binding cassette, subfamily B, member 1 (ABCB1) transporter, or P-glycoprotein, is an efflux protein implicated in the absorption and the distribution of various compounds, including tacrolimus and cyclosporine A. In vivo studies suggest an association between the ABCB1 1199G>A single nucleotide polymorphism (SNP) and tacrolimus intracellular accumulation. The aim of the present experimental study was to clarify in vitro the impact of the coding ABCB1 1199G>A SNP on ABCB1 transport activity towards both immunosuppressive drugs.
Two recombinant cell lines, i.e. Human Embryonic Kidney (HEK293) and Human Myelogenous Leukemia (K562) cells, overexpressing ABCB1 carrying either the wild-type allele (1199G) or its mutated counterpart (1199A), were generated. The impact of the 1199G>A SNP on ABCB1 activity towards rhodamine (Rh123), doxorubicin, vinblastine, tacrolimus and cyclosporine A was assessed by accumulation, cytotoxicity and/or kinetic experiments.
Tacrolimus accumulation was strongly decreased in cells overexpressing the wild-type protein (1199G) compared to control cells, confirming the ability of ABCB1 to transport tacrolimus. By contrast, overexpression of the variant protein (1199A) had nearly no effect on tacrolimus intracellular accumulation whatever the model used and the concentration tested. Unlike tacrolimus, our results also indicate that cyclosporine A, Rh123 and doxorubicin are transported in a similar extent by the wild-type and variant ABCB1 proteins while the variant protein seems to be more efficient for the transport of vinblastine.
ABCB1 encoded by the 1199G wild-type allele transports more efficiently tacrolimus in comparison to the 1199A variant protein. This observation indicates that the amino-acid substitution (Ser400Asn) encoded by the 1199A allele drastically decreases the ability of ABCB1 to drive the efflux of tacrolimus in a substrate-specific manner, in agreement with our previously published clinical data. Our study emphasizes the importance of the ABCB1 1199G>A polymorphism for ABCB1 activity and its potential to explain differences in drug response.
PMCID: PMC3951418  PMID: 24621983
10.  Metformin enhances tamoxifen-mediated tumor growth inhibition in ER-positive breast carcinoma 
BMC Cancer  2014;14:172.
Tamoxifen, an endocrine therapy drug used to treat breast cancer, is designed to interrupt estrogen signaling by blocking the estrogen receptor (ER). However, many ER-positive patients are low reactive or resistant to tamoxifen. Metformin is a widely used anti-diabetic drug with noteworthy anti-cancer effects. We investigated whether metformin has the additive effects with tamoxifen in ER-positive breast cancer therapy.
The efficacy of metformin alone and in combination with tamoxifen against ER-positive breast cancer was analyzed by cell survival, DNA replication activity, plate colony formation, soft-agar, flow cytometry, immunohistochemistry, and nude mice model assays. The involved signaling pathways were detected by western blot assay.
When metformin was combined with tamoxifen, the concentration of tamoxifen required for growth inhibition was substantially reduced. Moreover, metformin enhanced tamoxifen-mediated inhibition of proliferation, DNA replication activity, colony formation, soft-agar colony formation, and induction of apoptosis in ER-positive breast cancer cells. In addition, these tamoxifen-induced effects that were enhanced by metformin may be involved in the bax/bcl-2 apoptotic pathway and the AMPK/mTOR/p70S6 growth pathway. Finally, two-drug combination therapy significantly inhibited tumor growth in vivo.
The present work shows that metformin and tamoxifen additively inhibited the growth and augmented the apoptosis of ER-positive breast cancer cells. It provides leads for future research on this drug combination for the treatment of ER-positive breast cancer.
PMCID: PMC3976359  PMID: 24612549
Metformin; Tamoxifen; Estrogen receptor; Breast cancer
11.  Abnormal Default-Mode Network Homogeneity in First-Episode, Drug-Naive Major Depressive Disorder 
PLoS ONE  2014;9(3):e91102.
Default mode network (DMN) is one of the most commonly recognized resting-state networks in major depressive disorder (MDD). However, the homogeneity of this network in MDD is poorly understood. As such, this study was conducted to determine whether or not an abnormal network homogeneity (NH) of DMN is observed in patients with first-episode and drug-naive MDD.
Twenty-four first-episode drug-naive patients with MDD and twenty-four healthy control subjects participated in the study. NH and independent component analysis (ICA) methods were used to analyze data.
Depressed patients exhibited a significantly increased NH in the left dorsal medial prefrontal cortex (MPFC) and decreased NH in the right inferior temporal gyrus (ITG) compared with the healthy control subjects. Receiver operating characteristic curves (ROC) were analyzed and results revealed that the NH values of MPFC and ITG could be applied as candidate markers with relatively high sensitivity and specificity to distinguish patients from healthy control subjects. No correlation was observed between the NH values of the two regions and clinical variables.
Our findings suggested that an abnormal DMN homogeneity could be observed in MDD, which highlight the importance of the DMN in the pathophysiology of MDD.
PMCID: PMC3946684  PMID: 24609111
12.  Genetics of schizophrenia: What do we know? 
Current psychiatry  2013;12(3):24-33.
PMCID: PMC3892949  PMID: 24443645
13.  Identification of the involvement of LOXL4 in generation of keratocystic odontogenic tumors by RNA-Seq analysis 
Keratocystic odontogenic tumors (KCOT) are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density (MVD) than dentigerous cysts (DC) and normal oral mucosa. To identify genes in the stroma of KCOT involved in tumor development and progression, RNA sequencing (RNA-Seq) was performed using samples from KCOT and primary stromal fibroblasts isolated from gingival tissues. Seven candidate genes that possess a function potentially related to KCOT progression were selected and their expression levels were confirmed by quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay. Expression of lysyl oxidase-like 4 (LOXL4), the only candidate gene that encodes a secreted protein, was enhanced at both the mRNA and protein levels in KCOT stromal tissues and primary KCOT stromal fibroblasts compared to control tissues and primary fibroblasts (P<0.05). In vitro, high expression of LOXL4 could enhance proliferation and migration of the human umbilical vein endothelial cells (HUVECs). There was a significant, positive correlation between LOXL4 protein expression and MVD in stroma of KCOT and control tissues (r=0.882). These data suggest that abnormal expression of LOXL4 of KCOT may enhance angiogenesis in KCOT, which may help to promote the locally aggressive biological behavior of KCOT.
PMCID: PMC3967310  PMID: 24357854
angiogenesis; keratocystic odontogenic tumor; lysyl oxidase-like 4; RNA-sequencing; tumor stromal fibroblast
14.  Pseudo-ischaemic ECG in a patient with amyotrophic lateral sclerosis surviving for a decade 
BMJ Case Reports  2012;2012:bcr0120125543.
A 58-year-old female with no history of heart disease was admitted to our hospital for abnormal ECG mimicking myocardial ischaemia. The ECG revealed persistent T-wave inversion in almost all leads, especially in precordial leads V2–V6. The patient had no complaints of chest pain, chest distress, short of breath or other atypical myocardial ischaemia symptoms. She had a history of amyotrophic lateral sclerosis (ALS) with a disease course more than 20 years. Examinations help rule out other diseases causing persistent T-wave inversion. Importantly, cardiac catheterisation showed nearly normal coronary arteries that could rule out myocardial ischaemia. Accordingly, the authors presumed that the pseudo-ischaemic ECG was associated with ALS in this patient. The findings of the present case provide new evidence that autonomic nervous system may involve in the pathophysiological progress of ALS.
PMCID: PMC3291013  PMID: 22665549
15.  Coordination of Engineered Factors with TET1/2 Promotes Early-Stage Epigenetic Modification during Somatic Cell Reprogramming 
Stem Cell Reports  2014;2(3):253-261.
Somatic cell reprogramming toward induced pluripotent stem cells (iPSCs) holds great promise in future regenerative medicine. However, the reprogramming process mediated by the traditional defined factors (OSMK) is slow and extremely inefficient. Here, we develop a combination of modified reprogramming factors (OySyNyK) in which the transactivation domain of the Yes-associated protein is fused to defined factors and establish a highly efficient and rapid reprogramming system. We show that the efficiency of OySyNyK-induced iPSCs is up to 100-fold higher than the OSNK and the reprogramming by OySyNyK is very rapid and is initiated in 24 hr. We find that OySyNyK factors significantly increase Tet1 expression at the early stage and interact with Tet1/2 to promote reprogramming. Our studies not only establish a rapid and highly efficient iPSC reprogramming system but also uncover a mechanism by which engineered factors coordinate with TETs to regulate 5hmC-mediated epigenetic control.
Graphical Abstract
•A combination of modified reprogramming factors (OySyNyK) is developed•A highly efficient and rapid reprogramming system is established•TET1/2 proteins are involved in rapid iPSC induction by OySyNyK•OySyNyK factors coordinate with TET proteins to promote rapid reprogramming
The reprogramming process mediated by the traditional defined factors (OSMK) is slow and inefficient. Sun, Jin, Chen, and colleagues have developed a combination of modified reprogramming factors (OySyNyK) in which the transactivation domain of the Yes-associated protein is fused to defined factors and establish a highly efficient and rapid reprogramming system.
PMCID: PMC3964280  PMID: 24672749
16.  Largely Enhanced Single-molecule Fluorescence in Plasmonic Nanogaps formed by Hybrid Silver Nanostructures 
It has been suggested that narrow gaps between metallic nanostructures can be practical for producing large field enhancement. We design a hybrid silver nanostructure geometry in which fluorescent emitters are sandwiched between silver nanoparticles and silver island film (SIF). A desired number of polyelectrolyte layers are deposited on the SIF surface before the self-assembly of a second silver nanoparticle layer. Layer-by-layer configuration provides a well-defined dye position. It allows us to study the photophyical behaviors of fluorophores in the resulting gap at the single molecule level. The enhancement factor of a fluorophore located in the gap is much higher than those on silver surfaces alone and on glass. These effects may be used for increased detectability of single molecules bound to surfaces which contain metallic structures for either biophysical studies or high sensitivity assays.
PMCID: PMC3709438  PMID: 23373787
17.  Soft plasmons with stretchable spectroscopic response based on thermally patterned gold nanoparticles 
Scientific Reports  2014;4:4182.
Flexible photonic crystals are attractive devices owing to their multifold tunable parameters additionally introduced by soft substrates or by nanostructured, nano-doped, or nano-embedded soft matters. This not only extends significantly the intrinsic functions of photonic crystals, but also facilitates easy integration of the photonic crystal device into various optoelectronic and sensing systems. So far, flexible metallic photonic structures have been constructed on micrometer scales with complex fabrication procedures. Much simpler and more reproducible methods are expected to achieve such metamaterials in large scales and at low costs. In address to these challenges, we developed a straightforward approach to create soft plasmonic photonic crystals consisting of gold nanolines arranged on stretchable substrates with nanoscale periods, centimeter-scale areas, and high reproducibility using annealed gold nanoparticle colloids.
PMCID: PMC3933943  PMID: 24567008
18.  The Functional Influences of Common ABCB1 Genetic Variants on the Inhibition of P-glycoprotein by Antrodia cinnamomea Extracts 
PLoS ONE  2014;9(2):e89622.
Antrodia cinnamomea is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of A. cinnamomea (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of ABCB1 genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC50 concentration of 1.51±0.08 µg/mL through non-competitive inhibition. The IC50 concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56±0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications.
PMCID: PMC3934917  PMID: 24586917
19.  Elevated Levels of MYB30 in the Phloem Accelerate Flowering in Arabidopsis through the Regulation of FLOWERING LOCUS T 
PLoS ONE  2014;9(2):e89799.
In Arabidopsis thaliana, the R2R3 MYB-like transcription factor MYB30 is a positive regulator of the pathogen-induced hypersensitive response and of brassinosteroid and abscisic acid signaling. Here, we show that MYB30 expressed under the control of the strong phloem-specific SUC2 promoter accelerates flowering both in long and short days. Early flowering is mediated by elevated expression of FLOWERING LOCUS T (FT), which can be observed in the absence and presence of CONSTANS (CO), the main activator of FT. CO-independent activation by high MYB30 expression results in FT levels that remain below those observed in the wild-type plants, which show an additive CO-dependent activation. In contrast, TWIN SISTER OF FT (TSF) is repressed in plants expressing high levels of MYB30 in the phloem. In transient assays, MYB30 and CO additively increase the activity of a reporter construct driven by a 1 kb FT promoter. Acceleration of flowering by MYB30 does not require the presence of salicylic acid and is independent of FLC. Taken together, increased levels of MYB30, which was reported to be induced in response to the perception of pathogens, can accelerate flowering and MYB30 may thus be a candidate to mediate cross-talk between gene networks involved in biotic stress perception and flowering time.
PMCID: PMC3934951  PMID: 24587042
20.  Modulation of Anopheles stephensi Gene Expression by Nitroquine, an Antimalarial Drug against Plasmodium yoelii Infection in the Mosquito 
PLoS ONE  2014;9(2):e89473.
Antimalarial drugs may impact mosquito’s defense against Plasmodium parasites. Our previous study showed nitroquine significantly reduced infection of Anopheles stephensi by Plasmodium yoelii, but the underlying mechanism remains unclear. In order to understand how transmission capacity of An. stephensi was affected by nitroquine, we explored the transcriptome of adult females after different treatments, examined changes in gene expression profiles, and identified transcripts affected by the drug and parasite.
Methodology/Principal Findings
We extended massively parallel sequencing and data analysis (including gene discovery, expression profiling, and function prediction) to An. stephensi before and after Plasmodium infection with or without nitroquine treatment. Using numbers of reads assembled into specific contigs to calculate relative abundances (RAs), we categorized the assembled contigs into four groups according to the differences in RA values infection induced, infection suppressed, drug induced, and drug suppressed. We found both nitroquine in the blood meal and Plasmodium infection altered transcription of mosquito genes implicated in diverse processes, including pathogen recognition, signal transduction, prophenoloxidase activation, cytoskeleton assembling, cell adhesion, and oxidative stress. The differential gene expression may have promoted certain defense responses of An. stephensi against the parasite and decreased its infectivity.
Our study indicated that nitroquine may regulate several immune mechanisms at the level of gene transcription in the mosquito against Plasmodium infection. This highlights the need for better understanding of antimalarial drug’s impact on parasite survival and transmission. In addition, our data largely enriched the existing sequence information of An. stephensi, an epidemiologically important vector species.
PMCID: PMC3933544  PMID: 24586804
21.  Quantitative Analysis of the Anti-Proliferative Activity of Combinations of Selected Iron-Chelating Agents and Clinically Used Anti-Neoplastic Drugs 
PLoS ONE  2014;9(2):e88754.
Recent studies have demonstrated that several chelators possess marked potential as potent anti-neoplastic drugs and as agents that can ameliorate some of the adverse effects associated with standard chemotherapy. Anti-cancer treatment employs combinations of several drugs that have different mechanisms of action. However, data regarding the potential interactions between iron chelators and established chemotherapeutics are lacking. Using estrogen receptor-positive MCF-7 breast cancer cells, we explored the combined anti-proliferative potential of four iron chelators, namely: desferrioxamine (DFO), salicylaldehyde isonicotinoyl hydrazone (SIH), (E)-N′-[1-(2-hydroxy-5-nitrophenyl)ethyliden] isonicotinoyl hydrazone (NHAPI), and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), plus six selected anti-neoplastic drugs. These six agents are used for breast cancer treatment and include: paclitaxel, 5-fluorouracil, doxorubicin, methotrexate, tamoxifen and 4-hydroperoxycyclophosphamide (an active metabolite of cyclophosphamide). Our quantitative chelator-drug analyses were designed according to the Chou-Talalay method for drug combination assessment. All combinations of these agents yielded concentration-dependent, anti-proliferative effects. The hydrophilic siderophore, DFO, imposed antagonism when used in combination with all six anti-tumor agents and this antagonistic effect increased with increasing dose. Conversely, synergistic interactions were observed with combinations of the lipophilic chelators, NHAPI or Dp44mT, with doxorubicin and also the combinations of SIH, NHAPI or Dp44mT with tamoxifen. The combination of Dp44mT with anti-neoplastic agents was further enhanced following formation of its redox-active iron and especially copper complexes. The most potent combinations of Dp44mT and NHAPI with tamoxifen were confirmed as synergistic using another estrogen receptor-expressing breast cancer cell line, T47D, but not estrogen receptor-negative MDA-MB-231 cells. Furthermore, the synergy of NHAPI and tamoxifen was confirmed using MCF-7 cells by electrical impedance data, a mitochondrial inner membrane potential assay and cell cycle analyses. This is the first systematic investigation to quantitatively assess interactions between Fe chelators and standard chemotherapies using breast cancer cells. These studies are vital for their future clinical development.
PMCID: PMC3930662  PMID: 24586383
22.  Global Analysis of Lysine Acetylation Suggests the Involvement of Protein Acetylation in Diverse Biological Processes in Rice (Oryza sativa) 
PLoS ONE  2014;9(2):e89283.
Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the success of global analysis of lysine acetylation. A large number of proteins of diverse biological functions have been shown to be acetylated in several reports in human cells, E.coli, and dicot plants. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in monocots, particularly in the cereal crops. Here we report the mass spectrometric examination of lysine acetylation in rice (Oryza sativa). We identified 60 lysine acetylated sites on 44 proteins of diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated non-histone proteins. Examination of the amino acid composition revealed substantial amino acid bias around the acetylation sites and the amino acid preference is conserved among different organisms. Gene ontology analysis demonstrates that lysine acetylation occurs in diverse cytoplasmic, chloroplast and mitochondrial proteins in addition to the histone modifications. Our results suggest that lysine acetylation might constitute a regulatory mechanism for many proteins, including both histones and non-histone proteins of diverse biological functions.
PMCID: PMC3930695  PMID: 24586658
23.  Proteomic Analysis of the Ehrlichia chaffeensis Phagosome in Cultured DH82 Cells 
PLoS ONE  2014;9(2):e88461.
Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.
PMCID: PMC3928192  PMID: 24558391
24.  Selection of Suitable Reference Genes for Normalization of Quantitative Real-Time Polymerase Chain Reaction in Human Cartilage Endplate of the Lumbar Spine 
PLoS ONE  2014;9(2):e88892.
The quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used methods to study gene expression profiles, and it requires appropriate normalization for accurate and reliable results. Although several genes are commonly used as reference genes (such as GAPDH, ACTB, and 18S rRNA), they are also regulated and can be expressed at varying levels. In this study, we evaluated twelve well-known reference genes to identify the most suitable housekeeping gene for normalization of qRT-PCR in human lumbar vertebral endplate with Modic changes, by using the geNorm, NormFinder, and BestKeeper algorithms. Our results showed that the rarely-used SDHA was the most stable single reference gene, and a combination of three, SDHA, B2M, and LDHA, was the most suitable gene set for normalization in all samples. In addition, the commonly-used genes, GAPDH, ACTB and 18S rRNA, were all inappropriate as internal standards. The rankings of reference genes for the three types of Modic change differed, although SDHA and RPL13A uniformly ranked in the first and last position, respectively. Further simulated expression analysis validated that the arbitrary use of a reference gene could lead to the misinterpretation of data. Our study confirmed the necessity of exploring the expression stability of potential reference genes in each specific tissue and experimental situation before quantitative evaluation of gene expression by qRT-PCR.
PMCID: PMC3928306  PMID: 24558443
25.  Nitrogen Addition Significantly Affects Forest Litter Decomposition under High Levels of Ambient Nitrogen Deposition 
PLoS ONE  2014;9(2):e88752.
Forest litter decomposition is a major component of the global carbon (C) budget, and is greatly affected by the atmospheric nitrogen (N) deposition observed globally. However, the effects of N addition on forest litter decomposition, in ecosystems receiving increasingly higher levels of ambient N deposition, are poorly understood.
Methodology/Principal Findings
We conducted a two-year field experiment in five forests along the western edge of the Sichuan Basin in China, where atmospheric N deposition was up to 82–114 kg N ha–1 in the study sites. Four levels of N treatments were applied: (1) control (no N added), (2) low-N (50 kg N ha–1 year–1), (3) medium-N (150 kg N ha–1 year–1), and (4) high-N (300 kg N ha–1 year–1), N additions ranging from 40% to 370% of ambient N deposition. The decomposition processes of ten types of forest litters were then studied. Nitrogen additions significantly decreased the decomposition rates of six types of forest litters. N additions decreased forest litter decomposition, and the mass of residual litter was closely correlated to residual lignin during the decomposition process over the study period. The inhibitory effect of N addition on litter decomposition can be primarily explained by the inhibition of lignin decomposition by exogenous inorganic N. The overall decomposition rate of ten investigated substrates exhibited a significant negative linear relationship with initial tissue C/N and lignin/N, and significant positive relationships with initial tissue K and N concentrations; these relationships exhibited linear and logarithmic curves, respectively.
This study suggests that the expected progressive increases in N deposition may have a potential important impact on forest litter decomposition in the study area in the presence of high levels of ambient N deposition.
PMCID: PMC3925158  PMID: 24551152

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