Search tips
Search criteria

Results 1-22 (22)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
1.  Adipocytic Cells Augment the Support of Primitive Hematopoietic Cells In Vitro But Have No Effect in the Bone Marrow Niche Under Homeostatic Conditions 
Stem Cells and Development  2013;23(4):434-441.
Mesenchymal stem cells (MSCs), as well as osteoblastic cells derived from these MSCs, have been shown to be key components of the hematopoietic stem cell (HSC) niche. In this study, we wished to examine whether other cell types that are known to differentiate from MSCs similarly regulate the stem cell niche, namely cells of the adipocyte lineage. Recent studies have examined the role that adipocytes play in the biology of the HSCs in different bone locations and in transplantation settings; however, none have examined their role under homeostatic conditions. We compared the ability of adipocytic and nonadipocytic cell lines to support primitive hematopoietic cells in vitro. Preadipocytic cell lines demonstrated enhanced support of hematopoietic cells. Similarly, primary bone marrow (BM) cells treated with troglitazone, a drug that enhances adipogenesis, also demonstrated augmented support over control-treated stromal cells. We further examined the effects of increased adipocyte number in vivo under homeostatic conditions using troglitazone treatment and found that these alterations had no effect on HSC frequency. Taken together, we demonstrate that cells of the adipocyte lineage promote the ability of stromal cells to support primitive hematopoietic cells in vitro, yet alterations of adipocyte number and volume in vivo have no effect. These data suggest that adipocytes are not a component of the adult BM HSC niche under homeostatic conditions.
PMCID: PMC3996940  PMID: 24083324
2.  The Effect of Probiotics Supplementation on Helicobacter pylori Eradication Rates and Side Effects during Eradication Therapy: A Meta-Analysis 
PLoS ONE  2014;9(11):e111030.
Previous meta-analyses reported that probiotics improve the effectiveness of Helicobacter pylori (H. pylori) eradication during antibiotic therapy, while results regarding a possible reduction of side effects remained inconclusive. Moreover, the effectiveness of different strains of probiotics has not been studied so far. It is further conceivable that probiotics will produce additional effects only if antibiotics are relatively ineffective.
This meta-analysis includes eligible randomized controlled trials examining effects of probiotics supplementation on eradication rates (ER) and side effects, published up to May 2014. Sub-group analysis was performed to compare different probiotic strains and antibiotic therapies with different effectiveness in controls (ER <80% vs.>80%). Publication bias was assessed with funnel plots and Harbord's test. The quality of the trials was assessed with the Cochrane risk of bias tool.
Thirty-three RCTs involving a total of 4459 patients met the inclusion criteria in case of eradication rates of which 20 assessed total side effects in addition. Overall, the pooled eradication rate in probiotics supplementation groups was significantly higher than in controls (ITT analysis: RR 1.122, 95% CI 1.086–1.159, PP analysis: RR 1.114, 95% CI 1.070–1.159). Sub group-analysis could, however, confirm this finding only for four individual strains (Lactobacillus acidophilus, Lactobacillus casei DN-114001, Lactobacillus gasseri, and Bifidobacterium infantis 2036) and for relatively ineffective antibiotic therapies. There was a significant difference between groups in the overall incidence of side effects (RR 0.735, 95% CI 0.598–0.902). This result was, however, only confirmed for non-blinded trials.
The pooled data suggest that supplementation with specific strains of probiotics compared with eradication therapy may be considered an option for increasing eradication rates, particularly when antibiotic therapies are relatively ineffective. The impact on side effects remains unclear and more high quality trials on specific probiotic strains and side effects are thus needed.
PMCID: PMC4217763  PMID: 25365320
3.  The Sch9 Kinase Regulates Conidium Size, Stress Responses, and Pathogenesis in Fusarium graminearum 
PLoS ONE  2014;9(8):e105811.
Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.
PMCID: PMC4140829  PMID: 25144230
4.  Genome-Wide Characterization of Light-Regulated Genes in Neurospora crassa 
G3: Genes|Genomes|Genetics  2014;4(9):1731-1745.
The filamentous fungus Neurospora crassa responds to light in complex ways. To thoroughly study the transcriptional response of this organism to light, RNA-seq was used to analyze capped and polyadenylated mRNA prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120, and 240 min. More than three-quarters of all defined protein coding genes (79%) were expressed in these cells. The increased sensitivity of RNA-seq compared with previous microarray studies revealed that the RNA levels for 31% of expressed genes were affected two-fold or more by exposure to light. Additionally, a large class of mRNAs, enriched for transcripts specifying products involved in rRNA metabolism, showed decreased expression in response to light, indicating a heretofore undocumented effect of light on this pathway. Based on measured changes in mRNA levels, light generally increases cellular metabolism and at the same time causes significant oxidative stress to the organism. To deal with this stress, protective photopigments are made, antioxidants are produced, and genes involved in ribosome biogenesis are transiently repressed.
PMCID: PMC4169166  PMID: 25053707
Neurospora; RNA-seq; light
5.  Decreased miR-204 in H. pylori-Associated Gastric Cancer Promotes Cancer Cell Proliferation and Invasion by Targeting SOX4 
PLoS ONE  2014;9(7):e101457.
The molecular mechanism between Helicobacter pylori (H. pylori) infection and gastric cancer remained largely unknown. In this study, we determined the role of miRNA in H. pylori induced gastric cancer.
Methods and Results
We found that miR-204 was decreased in H. pylori positive tissues by qRT-PCR. Knockdown of miR-204 enhanced the invasion and proliferation ability of gastric cancer cells in vitro. Luciferase assay revealed that SOX4 was target gene of miR-204, which was found up-regulated in H. pylori positive tissues. Down-regulation of miR-204 and over-expression of SOX4 promoted epithelial-mesenchymal transition process.
Taken together, our findings demonstrated that miR-204 may act as a tumor suppressor in H. pylori induced gastric cancer by targeting SOX4.
PMCID: PMC4077842  PMID: 24984017
6.  Risk Factors and Prevalence of Helicobacter pylori Infection in Persistent High Incidence Area of Gastric Carcinoma in Yangzhong City 
Aim. The aim of this study was to investigate the prevalence and risk factors of H. pylori infection in areas with high prevalence of gastric cancer in Jiangsu Province, China. Methods. A prospective epidemiologic survey of H. pylori infection was accomplished in a natural population of 5417 individuals in Yangzhong city. Questionnaires and 13C-urea breath test for H. pylori infection were performed. Results. Among 5417 subjects who completed questionnaires and 13C-urea breath test, 3435 (63.41%) were H. pylori positive. The prevalence reached a peak at the age of 30–39 years (90.82%). There was significant difference between sexes and women had a higher infection rate than men. The prevalence of H. pylori infection was also associated with eating kipper food and fried food. No association between H. pylori prevalence and smoking or drinking was found. Compared to healthy individuals, people with dyspeptic diseases (peptic ulcer, gastroenteritis) presented a high prevalence of H. pylori infection. Using multivariate logistic regression analysis, age and history of peptic ulcer and gastroenteritis were the independent predictors for H. pylori infection. Conclusions. Yangzhong city had a high prevalence of H. pylori infection and was related to several risk factors. The underlying mechanisms are needed to be further investigated.
PMCID: PMC3914443  PMID: 24550981
7.  Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma 
Tumour Biology  2013;35(4):3755-3763.
Cytochrome b5 reductase 2 (CYB5R2), a member of the flavoprotein pyridine nucleotide cytochrome reductase family, is associated with a number of physiological reactions. However, its role in cancer, especially nasopharyngeal carcinoma (NPC), has not been addressed. Here, we investigate the transcript levels and promoter methylation status of CYB5R2 in NPC derived cell lines and tumor biopsies and experimentally address its role as a tumor suppressor gene. We find that CYB5R2 transcript levels are decreased in NPC cell lines and tumor biopsies. Promoter hypermethylation of CYB5R2 was detected in all six tested NPC cell lines and in 84 % of primary NPC tumor biopsies but not in normal nasopharyngeal epithelium. Clinically, CYB5R2 methylation was associated with lymph node metastasis in NPC patients (P < 0.05). The endogenous expression of CYB5R2 could be restored in vitro by the methyltransferase inhibitor 5-aza-2′-deoxycytidine in NPC cell lines. Ectopic expression of CYB5R2 had an inhibitory effect on proliferation, clonogenicity and migration of NPC cells. Moreover, in vivo tests in nude mice indicated that ectopic expression of CYB5R2 reduces the tumorigenicity of CYB5R2-negative NPC cells. Collectively, these findings suggest that CYB5R2 may be a functional tumor suppressor gene, frequently inactivated by hypermethylation of its promoter in NPC. We report here the first instance of epigenetic downregulation in NPC tumor biopsies of a key enzyme, CYB5R2, which is responsible for the detoxification of environmental carcinogens. We propose the possibility of utilizing CYB5R2 promoter methylation as a diagnostic biomarker of NPC in the future.
PMCID: PMC3980038  PMID: 24338690
CYB5R2; Tumor suppressor gene; Inactivation; Promoter hypermethylation; Nasopharyngeal carcinoma
8.  Practical Role of Mutation Analysis for Imatinib Treatment in Patients With Advanced Gastrointestinal Stromal Tumors: A Meta-Analysis 
PLoS ONE  2013;8(11):e79275.
Imatinib has become the standard first line treatment of gastrointestinal stromal tumors (GIST) in the advanced phase and adjuvant setting. We carried out an up-to-date meta-analysis to determine the practical role of mutation analysis for imatinib treatment in patients with advanced GIST.
Eligible studies were limited to imatinib treatment for patients with advanced GIST and reported on mutation analysis. Statistical analyses were conducted to calculate the odds ratio (OR), hazard ratio (HR) and 95% confidence interval (CI) using fixed-effects and random-effects models.
A total of 2834 patients from 3 randomized controlled trials and 12 cohort studies were included. The ORs of response rates in KIT exon 11-mutant GISTs were 3.504 (95% CI 2.549-4.816, p<0.001) and 3.521 (95% CI 1.731-7.165, p=0.001) compared with KIT exon 9-mutant and wild type GISTs, respectively. The HRs of progression-free survival in KIT exon 11-mutant GISTs were 0.365 (95% CI 0.301-0.444, p<0.001) and 0.375 (95% CI 0.270-0.519, p<0.001) compared with KIT exon 9-mutant and wild type GISTs. The HRs of overall survival in KIT exon 11-mutant GISTs were 0.388 (95% CI 0.293-0.515, p<0.001) and 0.400 (95% CI 0.297-0.538, p<0.001) compared with KIT exon 9-mutant and wild type GISTs. No statistical significant differences were found between KIT exon 9-mutant and wild type. The overall response rate in KIT-exon 11-mutant GISTs were 70.5% (65%-75.9%) compared with 57.1% (51%-63.2%) in KIT-positive GISTs. No evidence of publication bias was observed.
Patients with advanced GIST harboring a KIT exon 11 mutation have the best response rate and long-term survival with imatinib treatment. Mutation analysis would be more helpful than KIT expression analysis to decide appropriate therapy for a specific patient.
PMCID: PMC3817038  PMID: 24223922
9.  Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells 
PLoS ONE  2013;8(8):e67861.
Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.
PMCID: PMC3732288  PMID: 23936296
10.  A comparative proteomics study on matrix vesicles of osteoblast-like Saos-2 and U2-OS cells 
Matrix vesicles (MVs) play an important role in the initial stage of the process of bone mineralization, and are involved in multiple rare skeletal diseases with pathological mineralization or calcification. The aim of the study was to compare the proteomic profiling of osteoblast-like cells with and without mineralization ability (Saos-2 and U2-OS), and to identify novel mineralization-associated MV proteins. MVs were extracted using ExoQuick solution from mineralization-induced Saos-2 and U2-OS cells, and then were validated by transmission electron microscopy. A label-free quantitative proteomic method was used to compare the protein profiling of MVs from Saos-2 and U2-OS cells. Western-blots were used to confirm the expression of MVs proteins identified in proteomic studies. In our proteomic studies, we identified that 89 mineralization-related proteins were significantly up-regulated in Saos-2 MVs compared with U2-OS MVs. We further validated that two MVs proteins, protein kinase C α and ras-related protein Ral-A, were up-regulated in MVs of Saos-2 cells compared to those of U2-OS cells under mineralization-induction. Our findings suggest that protein kinase C α and ras-related protein Ral-A might be involved in bone mineralization as MVs components.
PMCID: PMC4204581  PMID: 25343104
Matrix vesicle; osteoblasts; mineralization; proteomics
11.  The FgHOG1 Pathway Regulates Hyphal Growth, Stress Responses, and Plant Infection in Fusarium graminearum 
PLoS ONE  2012;7(11):e49495.
Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley worldwide. In a previous study of systematic characterization of protein kinase genes in F. graminearum, mutants of three putative components of the osmoregulation MAP kinase pathway were found to have distinct colony morphology and hyphal growth defects on PDA plates. Because the osmoregulation pathway is not known to regulate aerial hyphal growth and branching, in this study we further characterized the functions of the FgHog1 pathway in growth, pathogenesis, and development. The Fghog1, Fgpbs2, and Fgssk2 mutants were all reduced in growth rate, aerial hyphal growth, and hyphal branching angle. These mutants were not only hypersensitive to osmotic stress but also had increased sensitivity to oxidative, cytoplasm membrane, and cell wall stresses. The activation of FgHog1 was blocked in the Fgpbs2 and Fgssk2 mutants, indicating the sequential activation of FgSsk2-FgPbs2-FgHog1 cascade. Interestingly, the FgHog1 MAPK pathway mutants appeared to be sensitive to certain compounds present in PDA. They were female sterile but retained male fertility. We also used the metabolomics profiling approach to identify compatible solutes that were accumulated in the wild type but not in the Fghog1 deletion mutant. Overall, our results indicate that the FgSsk2-FgPbs2-FgHog1 MAPK cascade is important for regulating hyphal growth, branching, plant infection, and hyperosmotic and general stress responses in F. graminearum.
PMCID: PMC3498113  PMID: 23166686
12.  The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae 
PLoS Pathogens  2012;8(9):e1002911.
Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M. oryzae. CAP1 may also play a role in feedback inhibition of Ras2 signaling when Pmk1 is activated.
Author Summary
In Magnaporthe oryzae, cAMP signaling is known to play an important role in surface recognition and plant penetration. The Mac1 adenylate cyclase is essential for plant infection. To better understand Mac1 activation mechanisms, in this study we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting protein is the adenylate cyclase associated protein (CAP) encoded by the CAP1 gene. Results from our study indicated that Cap1 is important for Mac1 activation and plant infection in M. oryzae. The Δcap1 mutant was defective in germ tube growth and appressorium formation and failed to cause typical blast lesions. Like LifeAct, Cap1 localized to apical patches in vegetative hyphae but not in invasive hyphae. The P2 proline-rich region was important for Cap1 localization but the actin-binding domain played a role in feedback inhibition of Ras signaling. To our knowledge, functional characterization of CAP genes has not been reported in filamentous fungi. Our results indicate that CAP1 is important for regulating adenylate cyclase activities, appressorium morphogenesis, and plant infection. Further characterization of CAP1 will be important to better understand the interaction between cAMP signaling and the PMK1 pathway in M. oryzae.
PMCID: PMC3435248  PMID: 22969430
13.  The AMT1 Arginine Methyltransferase Gene Is Important for Plant Infection and Normal Hyphal Growth in Fusarium graminearum 
PLoS ONE  2012;7(5):e38324.
Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.
PMCID: PMC3365026  PMID: 22693618
14.  Phosphate/Pyrophosphate and MV-related Proteins in Mineralisation: Discoveries from Mouse Models 
During the process of matrix vesicle (MV)-mediated initiation of mineralisation, chondrocytes and osteoblasts mineralise the extracellular matrix by promoting the seeding of basic calcium phosphate crystals of hydroxyapatite (HA) along the collagen fibrils. This orchestrated process is carefully regulated by the balanced action of propagators and inhibitors of calcification. The primary antagonistic regulators of extracellular matrix mineralisation are phosphate (Pi) and inorganic pyrophosphate (PPi). Studies in mouse models and in humans have established critical roles for Pi/PPi homeostasis in biomineralisation. In this review, we present the regulators of Pi/PPi, as derived from animal models, and discuss their clinical relevance to physiological and pathological mineralisation.
PMCID: PMC3372882  PMID: 22719218
Mineralisation; Matrix vesicles; PPi; Pi; MV-related proteins; OPN.
15.  MoSfl1 Is Important for Virulence and Heat Tolerance in Magnaporthe oryzae 
PLoS ONE  2011;6(5):e19951.
The formation of appressoria, specialized plant penetration structures of Magnaporthe oryzae, is regulated by the MST11-MST7-PMK1 MAP kinase cascade. One of its downstream transcription factor, MST12, is important for penetration and invasive growth but dispensable for appressorium formation. To identify additional downstream targets that are regulated by Pmk1, in this study we performed phosphorylation assays with a protein microarray composed of 573 M. oryzae transcription factor (TF) genes. Three of the TF genes phosphorylated by Pmk1 in vitro were further analyzed by coimmunoprecipitation assays. One of them, MoSFL1, was found to interact with Pmk1 in vivo. Like other Sfl1 orthologs, the MoSfl1 protein has the HSF-like domain. When expressed in yeast, MoSFL1 functionally complemented the flocculation defects of the sfl1 mutant. In M. oryzae, deletion of MoSFl1 resulted in a significant reduction in virulence on rice and barley seedlings. Consistent with this observation, the Mosfl1 mutant was defective in invasive growth in penetration assays with rice leaf sheaths. In comparison with that of vegetative hyphae, the expression level of MoSFL1 was increased in appressoria and infected rice leaves. The Mosfl1 mutant also had increased sensitivity to elevated temperatures. In CM cultures of the Mosfl1 and pmk1 mutants grown at 30°C, the production of aerial hyphae and melanization were reduced but their growth rate was not altered. When assayed by qRT-PCR, the transcription levels of the MoHSP30 and MoHSP98 genes were reduced 10- and 3-fold, respectively, in the Mosfl1 mutant. SFL1 orthologs are conserved in filamentous ascomycetes but none of them have been functionally characterized in non-Saccharomycetales fungi. MoSfl1 has one putative MAPK docking site and three putative MAPK phosphorylation sites. Therefore, it may be functionally related to Pmk1 in the regulation of invasive growth and stress responses in M. oryzae.
PMCID: PMC3098271  PMID: 21625508
16.  Aberrant methylation of secreted protein, acidic and rich in cysteine in human laryngeal and hypopharyngeal carcinoma 
Oncology Letters  2011;2(4):725-729.
Secreted protein, acidic and rich in cysteine (SPARC) has been found to be involved in various stages of tumor progression such as migration, invasion, extracellular matrix deposition and angiogenesis. To obtain an insight into the role of SPARC in the progression of laryngeal and hypopharyngeal carcinoma, we investigated SPARC transcription levels and promoter methylation in carcinoma cell lines and primary tumors. Reverse transcription-PCR showed that SPARC was silenced in laryngeal and hypopharyngeal carcinoma cell lines, in which aberrant promoter methylation was detected. Hypermethylation of SPARC was detected in 56.1% (23/41) of laryngeal carcinoma and 70.0% (7/10) of hypopharyngeal carcinoma biopsies, but only in 11.1% (1/9) of normal epithelial specimens by a methylation-specific PCR assay. Bisulphite genomic sequencing indicated that CpG sites in the SPARC promoter were heavily methylated in cell lines and primary tumors. Moreover, pharmacological demethylation treatment rescued SPARC expression with 5-aza-2′-deoxycytidine (5-aza-dC) in the laryngeal carcinoma cell lines. SPARC promoter hypermethylation was significantly correlated with lymph node metastasis (p<0.01). Our findings suggest that hypermethylation of SPARC is a frequent and tumor-specific event in laryngeal and hypopharyngeal carcinomas and may serve as a biomolecular marker for diagnosis and prognosis.
PMCID: PMC3406334  PMID: 22848256
DNA methylation; secreted protein; acidic and rich in cysteine; head and neck squamous cell carcinoma; tumor suppressor genes
17.  Multiple Plant Surface Signals are Sensed by Different Mechanisms in the Rice Blast Fungus for Appressorium Formation 
PLoS Pathogens  2011;7(1):e1001261.
Surface recognition and penetration are among the most critical plant infection processes in foliar pathogens. In Magnaporthe oryzae, the Pmk1 MAP kinase regulates appressorium formation and penetration. Its orthologs also are known to be required for various plant infection processes in other phytopathogenic fungi. Although a number of upstream components of this important pathway have been characterized, the upstream sensors for surface signals have not been well characterized. Pmk1 is orthologous to Kss1 in yeast that functions downstream from Msb2 and Sho1 for filamentous growth. Because of the conserved nature of the Pmk1 and Kss1 pathways and reduced expression of MoMSB2 in the pmk1 mutant, in this study we functionally characterized the MoMSB2 and MoSHO1 genes. Whereas the Momsb2 mutant was significantly reduced in appressorium formation and virulence, the Mosho1 mutant was only slightly reduced. The Mosho1 Momsb2 double mutant rarely formed appressoria on artificial hydrophobic surfaces, had a reduced Pmk1 phosphorylation level, and was nonresponsive to cutin monomers. However, it still formed appressoria and caused rare, restricted lesions on rice leaves. On artificial hydrophilic surfaces, leaf surface waxes and primary alcohols-but not paraffin waxes and alkanes- stimulated appressorium formation in the Mosho1 Momsb2 mutant, but more efficiently in the Momsb2 mutant. Furthermore, expression of a dominant active MST7 allele partially suppressed the defects of the Momsb2 mutant. These results indicate that, besides surface hydrophobicity and cutin monomers, primary alcohols, a major component of epicuticular leaf waxes in grasses, are recognized by M. oryzae as signals for appressorium formation. Our data also suggest that MoMsb2 and MoSho1 may have overlapping functions in recognizing various surface signals for Pmk1 activation and appressorium formation. While MoMsb2 is critical for sensing surface hydrophobicity and cutin monomers, MoSho1 may play a more important role in recognizing rice leaf waxes.
Author Summary
The rice blast fungus is a major pathogen of rice and a model for studying fungal-plant interactions. Like many other fungal pathogens, it can recognize physical and chemical signals present on the rice leaf surface and form a highly specialized infection structure known as appressorium. A well conserved signal transduction pathway involving the protein kinase gene PMK1 is known to regulate appressorium formation and plant penetration in this pathogen. However, it is not clear about the sensor genes that are involved in recognizing various plant surface signals. In this study we functionally characterize two putative sensor genes called MoMSB2 and MoSHO1. Genetic and biochemical analyses indicated that these two genes have overlapping functions in recognizing different physical and chemical signals present on the rice leaf surface for the activation of the Pmk1 pathway and appressorium formation. We found that primary alcohols, a major component of leaf waxes in grasses, can be recognized by the rice blast fungus as chemical cues. While MoMSB2 is critical for sensing hydrophobicity and precursors of cutin molecules of rice leaves, MoSHO1 appears to be more important than MoMSB2 for recognizing wax components.
PMCID: PMC3024261  PMID: 21283781
18.  Orthogonal test design for optimization of the extraction of polysaccharide from Paeonia sinjiangensis K.Y. Pan 
Pharmacognosy Magazine  2011;7(25):4-8.
Paeonia sinjiangensis K.Y. Pan is a perennial herb belonging to the ranunculaceae family and it is one of the most important crude drugs in traditional Chinese medicine. In this article, Paeonia sinjiangensis K.Y. Pan rich in polysaccharide is used as an experimental material.
Materials and Method:
Study the effects of proportion, temperature, times and time taken for the extraction yield of polysaccharide through a single-factor exploration. Then, through an orthogonal experiment (L9(3)4), it was investigated to get the best extraction conditions.
The results showed that the ratio of solvent to raw material, number of extractions and duration of extraction were the main variables that influenced the yields of extracts. The separation procedure of precipitation with alcohol and the purification from the removing proteins were deeply analyzed. Meanwhile the contents of polysaccharide were determined by anthrone colorimetry.
The highest yield was obtained when the ratio of solvent to raw material, number of extractions, and duration of extraction were 8:1, 2, and 1.5 h, respectively. The content of soluble polysaccharide is 51.57%.
PMCID: PMC3065155  PMID: 21472071
Anthrone colorimetry; extraction; orthogonal experiment; Paeonia sinjiangensis K.Y. Pan; polysaccharide
19.  Total polyphenolic (flavonoids) content and antioxidant capacity of different Ziziphora clinopodioides Lam. extracts 
Pharmacognosy Magazine  2011;7(25):65-68.
This paper investigates the total polyphenolic and flavonoid content as well as the antioxidant activity of Ziziphora clinopodioides Lam. extracts of different polarity.
Materials and Methods:
The total polyphenolic content was analysed using the Folin–Ciocalteu method. Total flavonoid content analysis was performed using the colorimetric method.
The total polyphenolic content of Z. clinopodioides is concentrated in parts of ethyl acetate (19.27%), chloroform (4.99%) and n-butanol extracts (3.94%) containing a small amount of the total polyphenolic content. The petroleum ether (0.23%) and ethanol extracts (1.64%) contain almost no polyphenolic content. The total flavonoid content of Z. clinopodioides is concentrated in parts of ethyl acetate (65.61%), chloroform (14.36%) and n-butanol extracts (10.76%) containing a small amount of the total polyphenolic content. The Z. clinopodioides Lam. ethyl acetate extract exhibits a good antioxidant activity.
Ethyl acetate extracts contain a large number of polyphenolic compounds (19.27%) and flavonoids (65.61%) owing to good antioxidant capacity.
PMCID: PMC3065160  PMID: 21472082
Antioxidant capacity; total flavonoids; total polyphenolic; Ziziphora clinopodioides Lam
20.  TFPI-2 is a putative tumor suppressor gene frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma 
BMC Cancer  2010;10:617.
Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC).
Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2.
TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration.
Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC.
PMCID: PMC2992524  PMID: 21062455
21.  Physicochemical evaluation and essential oil composition analysis of Hyssopus cuspidatus Boriss from Xinjiang, China 
Pharmacognosy Magazine  2010;6(24):278-281.
It is reported that the plant Hyssopus cuspidatus Boriss from Xinjiang has great value. This article deals with the detailed pharmacognostic evaluation of the crude drug H. cuspidatus Boriss.
Materials and Methods:
The essential oil of H. cuspidatus Boriss from Xinjiang, China, was extracted by the method of hydrodistillation and the chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry (GC–MS).
The yield of essential oil based on the dry weight of the plant was 0.6%(w/w). Fifty compounds accounting for 99.42% of the total oil were identified. The major components were oxygenated terpenes (66.33%), monoterpenes (26.14%), oxygenated sesquiterpenes (1.25%), and octane (1.85%).
Oxygenated terpenes were the main group of the compounds. The physicochemical parameters presented in this article may be proposed as parameters to establish the authenticity of H. cuspidatus Boriss and can possibly aid pharmacognostic and taxonomic species identification.
PMCID: PMC2992139  PMID: 21120028
Essential oil; GC-MS; Hyssopus cuspidatus Boriss; pharmacognosy
22.  Rapid determination of paeoniflorin from Paeonia sinjiang K. Y. Pan. by rapid resolution liquid chromatography 
Pharmacognosy Magazine  2010;6(22):98-101.
A rapid, effective, binary reverse phase rapid resolution liquid chromatographic method has been developed for the determination of Paeoniflorin extracted from Paeonia sinjiang K. Y. Pan. with short run time. RRLC separation was achieved by using Agilent (Zorbas XDB-C18 4.6 mm × 50 mm, 1.8 μm) column with mobile phase complosed of methanol and 0.05 mol/l potassium phosphate monobasic. Flow rate was at 1.0 ml/min. Retention time was at about 1.16 min. Recovery rate was 99.8% and relative standard deviation of three replicate samples tested was 1.8%. RRLC method has been applied to both in vitro studies of paeoniflorin formulation and drug estimation in biological samples.
PMCID: PMC2900069  PMID: 20668573
Paeonia sinjiang K. Y. Pan; paeoniflorin; rapid resolution liquid chromatography

Results 1-22 (22)