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1.  Characterizing heat shock protein 90 gene of Apolygus lucorum (Meyer-Dür) and its expression in response to different temperature and pesticide stresses 
Cell Stress & Chaperones  2014;19(5):725-739.
In this study, we cloned a full-length cDNA of heat shock protein (HSP) gene of Apolygus lucorum (Meyer-Dür) [AlHSP90, KC109781] and investigated its expression in response to temperature and pesticide stresses. The open reading frame (ORF) of AlHSP90 is 2,169 bp in length, encoding a 722 amino acid polypeptide with a predicted molecular weight of 82.99 kDa. Transcriptional and translational expression profiles of AlHSP90 under extreme temperature or pesticide stresses were examined by fluorescent real-time quantitative PCR and Western blot. Results showed that the expression profiles of AlHSP90 protein were in high agreement with those of AlHSP90 RNA and indicated that AlHSP90 was not only an important gene for A. lucorum adults in response to extremely high temperature, but also involved in the resistance or tolerance to cyhalothrin, imidacloprid, chlorpyrifos, and emamectin benzoate, especially for female adults to emamectin benzoate and for male adults to cyhalothrin. Transcriptional results of AlHSP90 also confirmed that AlHSP90 was an important gene involved in the resistance or tolerance to both temperature and pesticide stresses. In addition, our study also revealed that ∼24 °C may be the suitable temperature range for A. lucorum survival, which is also confirmed by the results of the expression of AlHSP90, the nymph mortality, and the intrinsic rate of increase (rm) when A. lucorum is reared at six different temperatures. Therefore, these studies are significant in elucidating the AlHSP90 in response to temperature and pesticide stresses and would provide guidance for A. lucorum management with different pesticides or temperatures in fields.
PMCID: PMC4147072  PMID: 24623316
A. lucorum; HSP90; Pesticide; Extreme temperature; Temperature stress; Expression files
2.  Evaluation of Fast Food Behavior in Pre-School Children and Parents Following a One-Year Intervention with Nutrition Education  
A community-based intervention study was conducted to assess a nutrition education intervention on western style fast food consumption among Chinese children and parents. Eight kindergartens from three district areas of Hefei City (a total of 1252 children aged 4–6 years and their parents) were randomly selected. Descriptive and analytical statistical methods were used to evaluate the baseline, midterm, and final western style fast food knowledge, attitude, and practice in both parents and children were used to identify and compare the knowledge, attitude, and practice in the parents and children. Parents and children were divided into “intervention” and “control” groups based on nutrition education status. Consumption of western style fast food at breakfast in Chinese children and parents is not high. The main reasons for this in children is that consumption of western style fast food is not viewed as “food”, but rather as a “gift” or “interesting”. The time of children’s consumption of western style fast food is mostly likely to be in the weekends. The nutrition education modified the parents’ western style fast food behavior (p < 0.01), although it did not change significantly in children. The healthy nutrition concept should be built up among Chinese, especially in children. Insights from the families provide leads for future research and ideas for the nutrition education.
PMCID: PMC4113844  PMID: 24983391
western style fast food; Chinese parents; young children
3.  Bundle Block Adjustment of Airborne Three-Line Array Imagery Based on Rotation Angles 
Sensors (Basel, Switzerland)  2014;14(5):8189-8202.
In the midst of the rapid developments in electronic instruments and remote sensing technologies, airborne three-line array sensors and their applications are being widely promoted and plentiful research related to data processing and high precision geo-referencing technologies is under way. The exterior orientation parameters (EOPs), which are measured by the integrated positioning and orientation system (POS) of airborne three-line sensors, however, have inevitable systematic errors, so the level of precision of direct geo-referencing is not sufficiently accurate for surveying and mapping applications. Consequently, a few ground control points are necessary to refine the exterior orientation parameters, and this paper will discuss bundle block adjustment models based on the systematic error compensation and the orientation image, considering the principle of an image sensor and the characteristics of the integrated POS. Unlike the models available in the literature, which mainly use a quaternion to represent the rotation matrix of exterior orientation, three rotation angles are directly used in order to effectively model and eliminate the systematic errors of the POS observations. Very good experimental results have been achieved with several real datasets that verify the correctness and effectiveness of the proposed adjustment models.
PMCID: PMC4063068  PMID: 24811075
airborne three-line array imagery; block adjustment; rotation angle; systematic error compensation; orientation image
4.  Immunoregulation effect of crude extract of C. elegans on allergic asthma 
To explore effects of natural crude extract of C. elegans on treatment of asthma. Method: Obtain crude extract of C. elegans from synchronically incubated C. elegans via centrifugation, washing and ultrasonic emulsification, etc.; measure C. elegans’s protein molecular weight via SDS-polyacrylamide gel electrophoresis (SDS-PAG electrophoresis); construct animal models of asthma with 6-8-week-old BALB/c female mice sensitized by chicken ovalbumin (OVA); conduct immunotherapy on animals with asthma with different doses of mixture of C. elegans and OVA (COM) respectively; take PBS buffer group and OVA group as control groups; conduct inspection of cell factors and differential count of cells in serum IgE, IgG1 and IgG2a antibodies and bronchoalveolar lavage fluid (BALF) via enzyme linked immunosorbent assay (ELISA); and incise lung tissue for pathology observation. Result: C. elegans’s protein molecular weight is about 50 kd. In bronchoalveolar lavage fluid (BALF) of OVA group, cell factors IL-5 and IL-13 are more than those in PBS buffer group, but IL-2 and IFN-γ are less than those in PBS buffer group; these differences are of statistical significance (P<0.05). Total cellular score and number of eosinophile granulocyte in BALF of OVA group are more than those in PBS buffer group (P<0.05), and the difference in serum IgE, IgG1 and IgG2a between these two groups is of statistical significance (P<0.05). For groups treatment by different doses of COM, cell factors IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF) are less than those in OVA group, but IL-2 and IFN-γ are more than those in OVA group; these differences are of statistical significance (P<0.05). Total cellular score and number of eosinophile granulocyte in BALF of COM treatment groups are less than those in OVA group (P<0.05); serum IgE and IgG1 less than those in OVA group, but IgG2a is more than that in OVA group; these differences are of statistical significance (P<0.05). Conclusion: The natural crude extract of C. elegans has immunoregulation to animals with asthma.
PMCID: PMC4057837  PMID: 24955158
Caenorhabditis elegans; crude extract; asthma; immunoregulation
5.  Lipopolysaccharide Induces Up-Regulation of TGF-α through HDAC2 in a Rat Model of Bronchopulmonary Dysplasia 
PLoS ONE  2014;9(3):e91083.
Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification with decreased alveolar number and increased airspace. Previous studies suggested that transforming growth factor-α (TGF-α) may contribute to arrested alveolar development in BPD. Histone deacetylases (HDACs) control cellular signaling and gene expression. HDAC2 is crucial for suppression of inflammatory gene expression. Here we investigated whether HDAC2 was involved in the arrest of alveolarization, as well as the ability of HDAC2 to regulate TGF-α expression in a rat model of BPD induced by intra-amniotic injection of lipopolysaccharide (LPS). Results showed that LPS exposure led to a suppression of both HDAC1 and HDAC2 expression and activity, induced TGF-α expression, and disrupted alveolar morphology. Mechanistic studies showed that overexpression of HDAC2, but not HDAC1, suppressed LPS-induced TGF-α expression. Moreover, the HDAC inhibitor TSA or downregulation of HDAC2 by siRNA both significantly increased TGF-α expression in cultured myofibroblasts. Finally, preservation of HDAC activity by theophylline treatment improved alveolar development and attenuated TGF-α release. Together, these findings indicate that attenuation of TGF-α-mediated effects in the lung by enhancing HDAC2 may have a therapeutic effect on treating BPD.
PMCID: PMC3942494  PMID: 24595367
6.  Pleural fluid prealbumin and C-reactive protein in the differential diagnosis of infectious and malignant pleural effusions 
Clinical history and physical examination are helpful in indicating the potential causes of pleural effusions (PEs). However, the accurate diagnosis and establishment of the causes of PE is an ongoing challenge in daily clinical practice. The primary aim of this study was to distinguish between infectious PE and malignant PE (MPE) by measuring two major acute phase response biomarkers: prealbumin (PA) and C-reactive protein (CRP). The study was a prospective trial involving 151 patients who were diagnosed with infectious PE or MPE. Patients with infectious PE were divided into two subgroups: tuberculous PE (TBPE) and parapneumonic PE (PNPE). A further 58 patients with PEs that showed no evidence of MPE, TBPE or PNPE were classified as the chronic non-specific PE (NSPE) group. Demographic characteristics and pleural fluids of the subjects were collected consecutively. The discriminative properties of pleural fluid routine biochemistries, and PA and CRP were evaluated. PA, CRP and classical fluid parameters were also applied to classify patients with infectious PE and MPE. Receiver operating characteristics (ROC) analysis established the cutoffs of PA and CRP for discriminating between groups. Pleural fluid PA levels were significantly higher in the MPE group (n=47) than in the infectious PE group (n=104). Pleural fluid CRP levels were significantly higher in the infectious PE group than in the MPE group. Pleural fluid PA levels were identified to be moderately negatively correlated with CRP levels in the MPE group, with a statistically significant correlation coefficient of −0.352. The ROC curve showed that the sensitivity and specificity of PA for the diagnosis of MPE were 0.851 and 0.548, respectively, at the cutoff of 28.3 mg/l. The area under the curve (AUC) was 0.784 (95% CI, 0.707–0.861). Using CRP as a diagnostic parameter resulted in an comparable AUC of 0.810 (95% CI, 0.736–0.885), at the cutoff of 35.2 mg/l. Combinations of PA and CRP resulted in incrementally discriminating values for MPE, with a sensitivity of 0.617 and a specificity of 0.903. The measurement of PA and CRP levels in pleural fluid may be a useful adjunctive test in PE, as a potential differentiator between infectious PE and MPE.
PMCID: PMC3961133  PMID: 24669233
prealbumin; C-reactive protein; pleural effusion
7.  Identification of the Plasma Metabolomics as Early Diagnostic Markers between Biliary Atresia and Neonatal Hepatitis Syndrome 
PLoS ONE  2014;9(1):e85694.
Early detection is the most effective way to improve the clinical outcome of biliary atresia (BA). Emerging metabolomics provides a powerful platform for discovering novel biomarkers and biochemical pathways to improve early diagnosis. The aim of this study is to find the potential biomarkers to distinguish BA from neonatal hepatitis syndrome (NHS) by using a metabolomics method. We comprehensively analyzed the serum metabolites in a total of 124 blood samples from patients with BA or neonatal hepatitis syndrome (NHS) and from normal individuals using advanced metabolomic approaches, and found that the levels of glutarylcarnitine (C5DC) significantly increased in the BA group while the levels of threonine (Thr) significantly rose in the NHS group comparing with the other groups. The levels of glutamic acid (Glu) in the BA group were significantly elevated compared to those in the NHS group, but still lower than the hyperbilirubinemia and normal controls. The levels of propionyl carnitine (C3), isovaleryl carnitine (C5) and glutamine (Gln) were reduced in the BA group compared to those in the NHS group, but still higher than the hyperbilirubinemia and normal controls. This study demonstrates the possibility of metabolomics as non-invasive biomarkers for the early detection of BA and also provides new insight into pathophysiologic mechanisms for BA.
PMCID: PMC3885726  PMID: 24416443
8.  Low-Density Lipoprotein Receptor-Related Protein 6 (LRP6) rs10845498 Polymorphism Is Associated with a Decreased Risk of Non-Small Cell Lung Cancer 
Objectives: Low-density lipoprotein receptor-related protein 6 (LRP6) modulates Wnt signaling transduction. Altered LRP6 expression leads to abnormal Wnt protein activation, cell proliferation and tumorigenesis. This study investigated the association between LRP6 single-nucleotide polymorphisms (SNPs) and non-small-cell lung cancer (NSCLC) in a Chinese population. Methods: A total of 500 NSCLC patients and 500 healthy controls were recruited for assessment of four LRP6 SNPs using the SEQUENOM MassARRAY matrix-assisted laser desorption ionization-time of flight mass spectrometry. The association between genotype and NSCLC risk was evaluated by computing the odds ratio (OR) and 95% confidence interval (CI) with multivariate unconditional logistic regression analyses. Results: The frequency of the LRP6 rs10845498 genotype was 60.9% (A/A), 35.5% (AG) and 3.6% (GG) in patients with lung squamous cell carcinoma (SCC) and 69.2% (A/A), 27.2% (A/G) and 3.6% (GG) in controls. Logistic regression analysis revealed that the LRP6 rs10845498 A/A major allele was associated with a reduced risk in developing lung SCC (OR = 0.69; 95% CI, 0.48-1.00; P=0.04), and tobacco smokers had a 2.21 fold greater risk in developing SCC than nonsmokers (p<0.01, 95% CI, 1.72-2.85), and tobacco smokers who carried an “A” allele (AA+AG) in rs6488507 had a 2.34-fold greater risk in developing NSCLC than other patients (p< 0.01, 95%CI, 1.74-3.13). Conclusions: The LRP6 rs10845498 SNP is associated with a reduced risk of lung SCC, while tobacco smoke increases the risk. LRP6 rs6488507 polymorphism synergistically increased the risk of NSCLC in tobacco smokers. Further studies are needed to elucidate the functional impact of LRP6 expression and activity in NSCLC.
PMCID: PMC4025167  PMID: 24843317
Non-small cell lung cancer; genetic susceptibility; low-density lipoprotein receptor-related protein 6; single nucleotide polymorphism.
9.  Eupafolin and Ethyl Acetate Fraction of Kalanchoe gracilis Stem Extract Show Potent Antiviral Activities against Enterovirus 71 and Coxsackievirus A16 
Enterovirus 71 (EV71) and coxsackievirus A16 (CoxA16) are main pathogens of hand-foot-and-mouth disease, occasionally causing aseptic meningitis and encephalitis in tropical and subtropical regions. Kalanchoe gracilis, Da-Huan-Hun, is a Chinese folk medicine for treating pain and inflammation, exhibiting antioxidant and anti-inflammatory activities. Our prior report (2012) cited K. gracilis leaf extract as moderately active against EV71 and CoxA16. This study further rates antienteroviral potential of K. gracilis stem (KGS) extract to identify potent antiviral fractions and components. The extract moderately inhibits viral cytopathicity and virus yield, as well as in vitro replication of EV71 (IC50 = 75.18 μg/mL) and CoxA16 (IC50 = 81.41 μg/mL). Ethyl acetate (EA) fraction of KGS extract showed greater antiviral activity than that of n-butanol or aqueous fraction: IC50 values of 4.21 μg/mL against EV71 and 9.08 μg/mL against CoxA16. HPLC analysis, UV-Vis absorption spectroscopy, and plaque reduction assay indicate that eupafolin is a vital component of EA fraction showing potent activity against EV71 (IC50 = 1.39 μM) and CoxA16 (IC50 = 5.24 μM). Eupafolin specifically lessened virus-induced upregulation of IL-6 and RANTES by inhibiting virus-induced ERK1/2, AP-1, and STAT3 signals. Anti-enteroviral potency of KGS EA fraction and eupafolin shows the clinical potential against EV71 and CoxA16 infection.
PMCID: PMC3775429  PMID: 24078828
10.  Epidermal Growth Factor Receptor Exon 20 Mutation Increased in Post-Chemotherapy Patients with Non-Small Cell Lung Cancer Detected with Patients' Blood Samples1 
Translational Oncology  2013;6(4):504-510.
PURPOSE: Patients with non-small cell lung cancer (NSCLC) and epidermal growth factor receptor (EGFR)-mutations have excellent response to EGFR tyrosine kinase inhibitors (TKIs), and exon 20 mutation accounts for most of TKI drug resistance. Nested polymerase chain reaction (PCR) was used to detect EGFR exon 20 mutations of patients with NSCLC after chemotherapy. The same is being analyzed with patients' characteristics. METHODS: Peripheral blood samples were collected from 273 patients with NSCLC, including 143 with adenocarcinoma (ADC) and 130 with squamous cell carcinoma (SCC), after chemotherapy. DNA was extracted from whole blood for nested PCR amplification and purification. Sequencing was carried out in an automated 3730 sequencer, followed by analysis of EGFR exon 20 mutations from nested PCR products. RESULTS: The mutations of EGFR exon 20 were mainly point mutations in rs1050171 (c.2361A>G) and rs56183713 (c.2457G>A). The point mutation was 28.21%, 28.46%, and 27.97% in patients with NSCLC, ADC and SCC, respectively. Men had an equivalent mutation (27.18%) to women (30.77%). The mutation in smokers and nonsmokers was 27.68% and 29.17%, respectively. In unselected patients, there was no correlation between EGFR exon 20 mutations and patients' characteristics of age, gender, smoking history, histologic type, or tumor-node-metastasis (TNM) staging system. In subgroup analyses, the EGFR mutation of patients with SCC was correlated with TNM stage [P = .013; odds ratio = 1.758; 95% confidence interval (CI) = 1.125–2.747]. CONCLUSIONS: The data indicate that the chemotherapy may induce EGFR-TKI-resistant mutation in NSCLC cells and EGFR-TKI should be used in the early stage of NSCLC but not after chemotherapy.
PMCID: PMC3730025  PMID: 23908693
11.  Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth, Plutella xyllostella 
PLoS ONE  2013;8(4):e62098.
Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic of pheromone receptors. In the Xenopus-based functional study and in situ hybridization, PxylOR4 is defined as another pheromone receptor in addition to the previously characterized PxylOR1. In the study of interaction between PRs and PBPs, PxylPBPs could increase the sensitivity of the complex expressing oocyte cells to the ligand pheromone component while decreasing the sensitivity to pheromone analogs. We deduce that activating pheromone receptors in olfactory receptor neurons requires some role of PBPs to pheromone/PBP complex. If the chemical signal is not the pheromone component, but instead, a pheromone analog with a similar structure, the complex would have a decreased ability to activate downstream pheromone receptors.
PMCID: PMC3633919  PMID: 23626773
12.  Polymorphisms of CHRNA5-CHRNA3-CHRNB4 Gene Cluster and NSCLC Risk in Chinese Population1 
Translational Oncology  2012;5(6):448-452.
AIM: To explore the potential association between single-nucleotide polymorphisms (SNPs) and haplotypes of the CHRNA5-CHRNA3-CHRNB4 gene cluster and the non-small cell lung cancer (NSCLC) susceptibility in never-smoking Chinese. METHODS: A case-control study was conducted with 200 NSCLC patients and 200 healthy controls, matched on age and sex. Five SNPs distributed in CHRNA5-CHRNA3-CHRNB4 gene cluster were selected for genotyping. The association between genotype and lung cancer risk was evaluated by computing the odds ratio (OR) and 95% confidence interval (CI) from multivariate unconditional logistic regression analyses with adjustment for gender and age. RESULTS: For CHRNA3 rs578776 status, data were available in 199 NSCLC patients and 199 controls. The G/G homozygote in CHRNB4 rs7178270 had a reduced risk of developing NSCLC (OR = 0.553; 95% CI = 0.309–0.989; P = .0437), especially squamous cell carcinoma (SQC) (OR = 0.344; 95% CI = 0.161–0.732; P = .0043), compared with those who carry at least one C allele (C/C and C/G). The polymorphisms of rs578776, rs938682, rs17486278, and rs11637635 were not significantly different between controls and cases or between controls and histologic subgroups, adenocarcinoma and SQC, respectively. CONCLUSIONS: In our study, we found that the SNP of CHRNB4 rs7178270 is significantly associated with reduced risk of NSCLC, especially with reduced risk of SQC in never-smoking Chinese population.
PMCID: PMC3567724  PMID: 23397474
13.  Candidate Olfaction Genes Identified within the Helicoverpa armigera Antennal Transcriptome 
PLoS ONE  2012;7(10):e48260.
Antennal olfaction is extremely important for insect survival, mediating key behaviors such as host preference, mate choice, and oviposition site selection. Multiple antennal proteins are involved in olfactory signal transduction pathways. Of these, odorant receptors (ORs) and ionotropic receptors (IRs) confer specificity on olfactory sensory neuron responses. In this study, we identified the olfactory gene repertoire of the economically important agricultural pest moth, Helicoverpa armigera, by assembling the adult male and female antennal transcriptomes. Within the male and female antennal transcriptomes we identified a total of 47 OR candidate genes containing 6 pheromone receptor candidates. Additionally, 12 IR genes as well as 26 odorant-binding proteins and 12 chemosensory proteins were annotated. Our results allow a systematic functional analysis across much of conventional ORs repertoire and newly reported IRs mediating the key olfaction-mediated behaviors of H. armigera.
PMCID: PMC3482190  PMID: 23110222
14.  Antiviral Ability of Kalanchoe gracilis Leaf Extract against Enterovirus 71 and Coxsackievirus A16 
Pandemic infection or reemergence of Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) occurs in tropical and subtropical regions, being associated with hand-foot-and-mouth disease, herpangina, aseptic meningitis, brain stem encephalitis, pulmonary edema, and paralysis. However, effective therapeutic drugs against EV71 and CVA16 are rare. Kalanchoe gracilis (L.) DC is used for the treatment of injuries, pain, and inflammation. This study investigated antiviral effects of K. gracilis leaf extract on EV71 and CVA16 replications. HPLC analysis with a C-18 reverse phase column showed fingerprint profiles of K. gracilis leaf extract had 15 chromatographic peaks. UV/vis absorption spectra revealed peaks 5, 12, and 15 as ferulic acid, quercetin, and kaempferol, respectively. K. gracilis leaf extract showed little cytotoxicity, but exhibited concentration-dependent antiviral activities including cytopathic effect, plaque, and virus yield reductions. K. gracilis leaf extract was shown to be more potent in antiviral activity than ferulic acid, quercetin, and kaempferol, significantly inhibiting in vitro replication of EV71 (IC50 = 35.88 μg/mL) and CVA16 (IC50 = 42.91 μg/mL). Moreover, K. gracilis leaf extract is a safe antienteroviral agent with the inactivation of viral 2A protease and reduction of IL-6 and RANTES expressions.
PMCID: PMC3361180  PMID: 22666293
15.  Correlations of circulating peptide YY and ghrelin with body weight, rate of weight gain, and time required to achieve the recommended daily intake in preterm infants 
The objective was to elucidate the relationships between serum concentrations of the gut hormone peptide YY (PYY) and ghrelin and growth development in infants for potential application to the clinical observation index. Serum concentrations of PYY and ghrelin were measured using radioimmunoassay from samples collected at the clinic. For each patient, gestational age, birth weight, time required to return to birth weight, rate of weight gain, time required to achieve recommended daily intake (RDI) standards, time required for full-gastric feeding, duration of hospitalization, and time of administration of total parenteral nutrition were recorded. Serum PYY and ghrelin concentrations were significantly higher in the preterm group (N = 20) than in the full-term group (N = 20; P < 0.01). Within the preterm infant group, the serum concentrations of PYY and ghrelin on postnatal day (PND) 7 (ghrelin = 1485.38 ± 409.24; PYY = 812.37 ± 153.77 ng/L) were significantly higher than on PND 1 (ghrelin = 956.85 ± 223.09; PYY = 545.27 ± 204.51 ng/L) or PND 3 (ghrelin = 1108.44 ± 351.36; PYY = 628.96 ± 235.63 ng/L; P < 0.01). Both serum PYY and ghrelin concentrations were negatively correlated with body weight, and the degree of correlation varied with age. Serum ghrelin concentration correlated negatively with birth weight and positively with the time required to achieve RDI (P < 0.05). In conclusion, serum PYY and ghrelin concentrations reflect a negative energy balance, predict postnatal growth, and enable compensation. Further studies are required to elucidate the precise concentration and roles of PYY and ghrelin in newborns and to determine the usefulness of measuring these hormones in clinical practice.
PMCID: PMC3854269  PMID: 22527125
Peptide YY; Ghrelin; Preterm infants; Recommended daily intake
16.  Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium anisopliae and M. acridum 
PLoS Genetics  2011;7(1):e1001264.
Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ∼30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ∼16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogenous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.
Author Summary
Aside from playing a crucial role in natural ecosystems, entomopathogenic fungi are being developed as environmentally friendly alternatives for the control of insect pests. We conducted the first genomic study of two of the best characterized entomopathogens, Metarhizium anisopliae and M. acridum. M. anisopliae is a ubiquitous pathogen of >200 insect species and a plant growth promoting colonizer of rhizospheres. M. acridum is a specific pathogen of locusts. Important findings of this study included: 1) Both M. anisopliae and M. acridum have a very large number of genes encoding secreted proteins, and many of these play roles in fungus-insect interactions. 2) M. anisopliae has more genes than M. acridum, which may be associated with adaptation to multiple insect hosts. 3) Unlike M. acridum, the M. anisopliae genome contains many more transposase genes and shows no evidence of repeat-induced point mutations. The lack of repeat-induced mutations may have allowed the lineage-specific gene duplications that have contributed to its adaptability. 4) High-throughput transcriptomics identified the strategies by which these fungi overcome their insect hosts and achieve specificity. These genome sequences will provide the basis for a comprehensive understanding of fungal–plant–insect interactions and will contribute to our understanding of fungal evolution and ecology.
PMCID: PMC3017113  PMID: 21253567
17.  Requirement of a Mitogen-Activated Protein Kinase for Appressorium Formation and Penetration of Insect Cuticle by the Entomopathogenic Fungus Beauveria bassiana▿ †  
Beauveria bassiana is an important insect-pathogenic fungus that invades insects by direct penetration of the host cuticle. To delineate the molecular mechanisms involved in fungal infection, a mitogen-activated protein kinase (MAPK) gene, Bbmpk1, which encodes a YERK1 family MAPK was isolated and characterized. Targeted gene disruption of Bbmpk1 resulted in a complete loss of virulence when applied topically to host insects but did not affect growth of the fungus when conidia were injected directly into the hemocoel. Hyphae of the mutant strain growing in the insect hemocoel were unable to penetrate the cuticle growing outwards and consequently failed to sporulate on the cadaver surface. These data suggest that BbMPK1 is essential for penetration of the insect cuticle both from the outside and from the inside-out in order to escape and disperse from the host. Inactivation of BbMPK1 also caused a significant decrease in fungal adhesion to insect cuticles and eliminated their ability to form appressoria. In order to identify downstream genes regulated by BbMPK1, a suppressive subtractive hybridization (SSH) library was generated comparing mutant and wild-type transcripts isolated during appressorium formation. Thirty-one genes screened from the SSH library were determined to be expressed in the wild-type strain but either significantly reduced or not expressed in the mutant. Ten genes showed high or medium similarity to known protein encoding genes, including proteins involved in cell surface hydrophobicity, lipid metabolism, microtubule dynamics, mitochondrial electron transport, chromatin remodeling, transcription, rRNA processing, small nucleolar RNA accumulation, oxidation of aldehydes, translation, and likely other cellular processes.
PMCID: PMC2849248  PMID: 20139313
18.  Reactions of (Trialkylsilyl)vinylketenes with Lithium Ynolates: A New Benzannulation Strategy 
Organic letters  2005;7(18):3905-3908.
(Trialkylsilyl)vinylketenes react with lithium ynolates to produce highly substituted phenols in a new benzannulation strategy that proceeds via the 6π electrocyclization of an intermediate 3-(oxido)dienylketene.
PMCID: PMC2897055  PMID: 16119928
19.  Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence▿  
Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ΔBbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.
PMCID: PMC2798667  PMID: 19854926
20.  Mitogen-Activated Protein Kinase hog1 in the Entomopathogenic Fungus Beauveria bassiana Regulates Environmental Stress Responses and Virulence to Insects▿ †  
Applied and Environmental Microbiology  2009;75(11):3787-3795.
Beauveria bassiana is an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. However, its insecticide efficacy in the field is often influenced by adverse environmental factors. Thus, understanding the genetic regulatory processes involved in the response to environmental stress would facilitate engineering and production of a more efficient biocontrol agent. Here, a mitogen-activated protein kinase (MAPK)-encoding gene, Bbhog1, was isolated from B. bassiana and shown to encode a functional homolog of yeast HIGH-OSMOLARITY GLYCEROL 1 (HOG1). A Bbhog1 null mutation was generated in B. bassiana by targeted gene replacement, and the resulting mutants were more sensitive to hyperosmotic stress, high temperature, and oxidative stress than the wild-type controls. These results demonstrate the conserved function of HOG1 MAPKs in the regulation of abiotic stress responses. Interestingly, ΔBbhog1 mutants exhibited greatly reduced pathogenicity, most likely due to a decrease in spore viability, a reduced ability to attach to insect cuticle, and a reduction in appressorium formation. The transcript levels of two hydrophobin-encoding genes, hyd1 and hyd2, were dramatically decreased in a ΔBbhog1 mutant, suggesting that Bbhog1 may regulate the expression of the gene associated with hydrophobicity or adherence.
PMCID: PMC2687298  PMID: 19363067
21.  Reversion-induced LIM interaction with Src reveals a novel Src inactivation cycle 
The Journal of Cell Biology  2009;184(6):785-792.
Aberrant Src activation plays prominent roles in cancer progression. However, how Src is activated in cancer cells is largely unknown. Genetic Src-activating mutations are rare and, therefore, are insufficient to account for Src activation commonly found in human cancers. In this study, we show that reversion-induced LIM (RIL), which is frequently lost in colon and other cancers as a result of epigenetic silencing, suppresses Src activation. Mechanistically, RIL suppresses Src activation through interacting with Src and PTPL1, allowing PTPL1-dependent dephosphorylation of Src at the activation loop. Importantly, the binding of RIL to Src is drastically reduced upon Src inactivation. Our results reveal a novel Src inactivation cycle in which RIL preferentially recognizes active Src and facilitates PTPL1-mediated inactivation of Src. Inactivation of Src, in turn, promotes dissociation of RIL from Src, allowing the initiation of a new Src inactivation cycle. Epigenetic silencing of RIL breaks this Src inactivation cycle and thereby contributes to aberrant Src activation in human cancers.
PMCID: PMC2699145  PMID: 19307596
22.  Tsg101 can replace Nedd4 function in ASV Gag release but not membrane targeting 
Virology  2008;377(1):30-38.
The Late (L) domain of the avian sarcoma virus (ASV) Gag protein binds Nedd4 ubiquitin ligase E3 family members and is the determinant of efficient virus release in avian and mammalian cells. We previously demonstrated that Nedd4 and Tsg101 constitutively interact raising the possibility that Nedd4 links ASV Gag to the ESCRT machinery. We now demonstrate that covalently linking Tsg101 to ASV Gag lacking the Nedd4 binding site (Δp2b-Tsg101) ablates the requirement for Nedd4, but the rescue of budding occurs by use of a different budding mechanism than that used by wild type ASV Gag. The evidence that Tsg101 and Nedd4 direct release by different pathways is: (i) Release of the virus-like particles (VLPs) assembled from Gag in DF-1, an avian cell line, was resistant to dominant-negative interference by a Tsg101 mutant previously shown to inhibit release of both HIV and Mo-MLV. (ii) Release of VLPs from DF-1 cells was resistant to siRNA-mediated depletion of the endogenous pool of Tsg101 in these cells. (iii) VLPs assembled from wild-type ASV Gag exhibited highly efficient release from endosome-like membrane domains enriched in the tetraspanin protein CD63 or a fluorescent analogue of the phospholipid phosphatidylethanolamine. However, the VLPs assembled from the L domain mutant Δp2b or a chimeric Δp2b-Tsg101 Gag lacked these domain markers even though the chimeric Gag was released efficiently compared to the Δp2b mutant. These results suggest that Tsg101 and Nedd4 facilitate Gag release through functionally exchangeable but independent routes and that Tsg101 can replace Nedd4 function in facilitating budding but not directing through the same membranes.
PMCID: PMC2528022  PMID: 18555885
23.  The Functionally Exchangeable L Domains in RSV and HIV-1 Gag Direct Particle Release Through Pathways Linked by Tsg101 
Traffic (Copenhagen, Denmark)  2005;6(10):880-894.
The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101–Nedd4 interaction.
PMCID: PMC2692930  PMID: 16138902
Gag; HIV-1; L domain; Nedd4; RSV; Tsg101; Vps4
24.  A benzannulation strategy for the synthesis of phenols and heteroaromatic compounds based on the reaction of (trialkylsilyl)vinylketenes with lithium ynolates 
Tetrahedron  2008;64(5):915-925.
(Trialkylsilyl)vinylketenes react with lithium ynolates to generate 3-(oxido)dienylketenes which undergo rapid 6π-electrocyclization. The ultimate products of this benzannulation are highly substituted resorcinol monosilyl ethers which are formed via a [1,3] carbon to oxygen silyl group shift. Further transformations of the benzannulation products are described providing efficient access to ortho-benzoquinones and benzofuran, benzoxepine, and benzoxocine ring systems.
PMCID: PMC2350204  PMID: 19180173
(Trialkylsilyl)vinylketenes; Benzannulation; Ynolates; Electrocyclic ring closure
25.  Synthesis and Tribological Properties of WSe2 Nanorods 
Nanoscale Research Letters  2008;3(12):481-485.
The WSe2 nanorods were synthesized via solid-state reaction method and characterized by X-ray diffractometer, TEM, and HRTEM. The results indicated the WSe2 compounds had rod-like structures with diameters of 10–50 nm and lengths of 100–400 nm, and the growth process of WSe2 nanorods was discussed on the basis of the experimental facts. The tribological properties of WSe2 nanorods as additives in HVI500 base oil were investigated by UMT-2 multispecimen tribotester. Under the determinate conditions, the friction coefficient of the base oil containing WSe2 nanorods was lower than that of the base oil, and decreased with increasing mass fraction of WSe2 nanorods when it was <7 wt.%. Moreover, the base oil with the additives was rather suited to high load and high rotating speed. A combination of rolling friction, sliding friction, and stable tribofilm on the rubbing surface could explain the good friction and wear properties of WSe2 nanorods as additives.
PMCID: PMC2893792  PMID: 20592963
WSe2 nanorods; Growth mechanism; Lubrication additive; Tribological properties; Rotating speed

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