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1.  Molecular Characterization of Vitellogenin and Its Receptor Genes from Citrus Red Mite, Panonychus citri (McGregor) 
The production and uptake of yolk protein play an important role in the reproduction of all oviparous organisms. Vitellogenin (Vg) is the precursor of vitellin (Vn), which is the major egg storage protein, and vitellogenin receptor (VgR) is a necessary protein for the uptake of Vg into developing oocytes. In this paper, we characterize the full-length Vg and VgR, PcVg1 and PcVgR, respectively, of the citrus red mite Panonychus citri (McGregor). The PcVg1 cDNA is 5748 nucleotides (nt) with a 5553-nt open reading frame (ORF) coding for 1851 amino acids (aa), and the PcVgR is 6090 nt, containing an intact ORF of 5673 nt coding an expected protein of 1891 aa. The PcVg1 aa sequence shows a typical GLCG domain and several K/RXXR cleavage sites, and PcVgR comprises two ligand-binding domains, two epidermal growth factor (EGF)-like regions containing YWTD motifs, a transmembrane domain, and a cytoplasmic domain. An analysis of the aa sequences and phylogenetics implied that both genes were genetically distinct from those of ticks and insects. The transcriptional profiles determined by real-time quantitative PCR in different developmental stages showed that both genes present the same expressional tendencies in eggs, larvae, nymphs, and adults. This suggested that the biosynthesis and uptake of PcVg occurs coordinately. The strong reproductive capacity of P. citri has been hypothesized as an important factor in its resistance; consequently, understanding the molecular mechanisms regulating Vg and VgR are fundamental for mite control.
PMCID: PMC4394447  PMID: 25739087
Panonychus citri; vitellogenin; vitellogenin receptor; cloning; relative expression
2.  Oxidative Responses Induced by Pharmacologic Vitreolysis and/or Long-term Hyperoxia Treatment in Rat Lenses 
Current eye research  2013;38(6):639-648.
The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo.
Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors.
No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis.
Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.
PMCID: PMC3740155  PMID: 23534693
Cataract; hyperoxia; oxidative stress; pharmacologic vitreolysis; posterior vitreous detachment
3.  Exposure to Diflubenzuron Results in an Up-Regulation of a Chitin Synthase 1 Gene in Citrus Red Mite, Panonychus citri (Acari: Tetranychidae) 
Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB.
PMCID: PMC3975363  PMID: 24590130
Panonychus citri; chitin synthase 1; diflubenzuron; insect growth regulators; pest control
4.  Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila 
Mechanisms of development  2012;129(0):98-108.
Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.
PMCID: PMC3917606  PMID: 22677792
Hedgehog signaling; Costal-2; Patched; Non-cell autonomous overgrowth
5.  Identification and Characterization of Seven Glutathione S-Transferase Genes from Citrus Red Mite, Panonychus citri (McGregor) 
The citrus red mite, Panonychus citri (McGregor), is a global citrus pest, and has developed severe resistance to several types of acaricides. However, the molecular mechanisms of resistance in this mite remain unknown. In this study, seven full-length cDNAs encoding glutathione S-transferases (GSTs) genes were identified and characterized in P. citri. The effects of pyridaben and fenpropathrin exposure on the expression of these genes were also investigated. Phylogenetic analysis revealed that the seven GSTs genes in P. citri cloned in this study belong to three different cytosolic classes, including four in mu, two in delta and one in zeta. Among these seven GSTs genes, the relative expression level of PcGSTm1 was significantly higher in adult than in the other life stages (egg, larvae and nymph). Compared with the control, the mRNA levels of the seven GST genes did not change significantly following exposure to pyridaben at LC10. However, RT-qPCR results showed that, when exposed to LC10 of fenpropathrin, six GSTs gene (PcGSTm1, PcGSTm3, PcGSTm4, PcGSTd1, PcGSTd2 and PcGSTz1) transcripts increased in a time-dependent manner. This is the first insight into the molecular characteristics of GSTs gene cDNAs in P. citri. The elevated GSTs gene transcripts following exposure to fenpropathrin might be one of the mechanisms involved in detoxification of this acaricide.
PMCID: PMC3876109  PMID: 24351815
Panonychus citri; glutathione S-transferase; gene expression; pyridaben; fenpropathrin
6.  Mining Genes Involved in Insecticide Resistance of Liposcelis bostrychophila Badonnel by Transcriptome and Expression Profile Analysis 
PLoS ONE  2013;8(11):e79878.
Recent studies indicate that infestations of psocids pose a new risk for global food security. Among the psocids species, Liposcelis bostrychophila Badonnel has gained recognition in importance because of its parthenogenic reproduction, rapid adaptation, and increased worldwide distribution. To date, the molecular data available for L. bostrychophila is largely limited to genes identified through homology. Also, no transcriptome data relevant to psocids infection is available.
Methodology and Principal Findings
In this study, we generated de novo assembly of L. bostrychophila transcriptome performed through the short read sequencing technology (Illumina). In a single run, we obtained more than 51 million sequencing reads that were assembled into 60,012 unigenes (mean size = 711 bp) by Trinity. The transcriptome sequences from different developmental stages of L. bostrychophila including egg, nymph and adult were annotated with non-redundant (Nr) protein database, gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). The analysis revealed three major enzyme families involved in insecticide metabolism as differentially expressed in the L. bostrychophila transcriptome. A total of 49 P450-, 31 GST- and 21 CES-specific genes representing the three enzyme families were identified. Besides, 16 transcripts were identified to contain target site sequences of resistance genes. Furthermore, we profiled gene expression patterns upon insecticide (malathion and deltamethrin) exposure using the tag-based digital gene expression (DGE) method.
The L. bostrychophila transcriptome and DGE data provide gene expression data that would further our understanding of molecular mechanisms in psocids. In particular, the findings of this investigation will facilitate identification of genes involved in insecticide resistance and designing of new compounds for control of psocids.
PMCID: PMC3835895  PMID: 24278202
7.  De Novo Assembly, Gene Annotation, and Marker Discovery in Stored-Product Pest Liposcelis entomophila (Enderlein) Using Transcriptome Sequences 
PLoS ONE  2013;8(11):e80046.
As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels.
Methodology/Principal Findings
We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61%) unigenes were matched to known proteins in the NCBI non-redundant (Nr) protein database. These unigenes were further functionally annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST) genes, 19 putative carboxyl/cholinesterase (CCE) genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp) genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future.
We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying insecticide resistance or environmental stress, and will facilitate studies on population genetics for psocids, as well as providing useful information for functional genomic research in the future.
PMCID: PMC3828239  PMID: 24244605
8.  Utilization of PET-CT in target volume delineation for three-dimensional conformal radiotherapy in patients with non-small cell lung cancer and atelectasis 
To investigate the utilization of PET-CT in target volume delineation for three-dimensional conformal radiotherapy in patients with non-small cell lung cancer (NSCLC) and atelectasis.
Thirty NSCLC patients who underwent radical radiotherapy from August 2010 to March 2012 were included in this study. All patients were pathologically confirmed to have atelectasis by imaging examination. PET-CT scanning was performed in these patients. According to the PET-CT scan results, the gross tumor volume (GTV) and organs at risk (OARs, including the lungs, heart, esophagus and spinal cord) were delineated separately both on CT and PET-CT images. The clinical target volume (CTV) was defined as the GTV plus a margin of 6-8 mm, and the planning target volume (PTV) as the GTV plus a margin of 10-15mm. An experienced physician was responsible for designing treatment plans PlanCT and PlanPET-CT on CT image sets. 95% of the PTV was encompassed by the 90% isodose curve, and the two treatment plans kept the same beam direction, beam number, gantry angle, and position of the multi-leaf collimator as much as possible. The GTV was compared using a target delineation system, and doses distributions to OARs were compared on the basis of dose-volume histogram (DVH) parameters.
The GTVCT and GTVPET-CT had varying degrees of change in all 30 patients, and the changes in the GTVCT and GTVPET-CT exceeded 25% in 12 (40%) patients. The GTVPET-CT decreased in varying degrees compared to the GTVCT in 22 patients. Their median GTVPET-CT and median GTVPET-CT were 111.4 cm3 (range, 37.8 cm3-188.7 cm3) and 155.1 cm3 (range, 76.2 cm3-301.0 cm3), respectively, and the former was 43.7 cm3 (28.2%) less than the latter. The GTVPET-CT increased in varying degrees compared to the GTVCT in 8 patients. Their median GTVPET-CT and median GTVPET-CT were 144.7 cm3 (range, 125.4 cm3-178.7 cm3) and 125.8 cm3 (range, 105.6 cm3-153.5 cm3), respectively, and the former was 18.9 cm3 (15.0%) greater than the latter. Compared to PlanCT parameters, PlanPET-CT parameters showed varying degrees of changes. The changes in lung V20, V30, esophageal V50 and V55 were statistically significant (Ps< 0.05 for all), while the differences in mean lung dose, lung V5, V10, V15, heart V30, mean esophageal dose, esophagus Dmax, and spinal cord Dmax were not significant (Ps> 0.05 for all).
PET-CT allows a better distinction between the collapsed lung tissue and tumor tissue, improving the accuracy of radiotherapy target delineation, and reducing radiation damage to the surrounding OARs in NSCLC patients with atelectasis.
PMCID: PMC3608960  PMID: 23506629
Atelectasis; PET-CT; Non-small cell lung cancer; Target volume; Three-dimensional conformal radiotherapy
9.  A New Secondary Model Developed for the Growth Rate of Escherichia coli O157:H7 in Broth 
Indian Journal of Microbiology  2011;52(1):99-101.
This study was attempted to develop a new exponential sum model to describe the effect of temperature on growth rate (GR) of Escherichia coli O157:H7 in broth. The growth rates of E. coli O157:H7 at different storage temperatures (4, 10, 15, 20, 25, 30, and 35°C) estimated by fitting with the modified Gompertz model were used to develop secondary models such as square root model, Ratkowsky model and exponential sum model. Measures of coefficient of determination (R2), root mean square error (RMSE) and the sum of squares due to error (SSE) were employed to compare the performances of these three secondary models. Based on these criteria, the developed exponential sum model showed the better goodness-of-fit and performance.
PMCID: PMC3298592  PMID: 23449380
Escherichia coli O157:H7; Predictive microbiology; Exponential sum model; Broth
10.  Hydrogen saline prevents selenite-induced cataract in rats 
Molecular Vision  2013;19:1684-1693.
The aim of this study was to investigate the potential antioxidative effect and mechanism for the protective effects of hydrogen saline on selenite-induced cataract in rats.
Sprague-Dawley rat pups were divided into the following groups: control (Group A), selenite induced (Group B), and selenite plus hydrogen saline treated (Group C). Rat pups in Groups B and C received a single subcutaneous injection of sodium selenite (25 μmol/kg bodyweight) on postnatal day 12. Group C also received an intraperitoneal injection of H2 saline (5 ml/kg bodyweight) daily from postnatal day 8 to postnatal day 17. The development of cataract was assessed weekly by slit-lamp examination for 2 weeks. After sacrifice, extricated lenses were analyzed for activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, levels of malondialdehyde, reduced glutathione (GSH), and total sulfhydryl contents.
The magnitude of lens opacification in Group B was significantly higher than in Group A (p<0.05), while Group C had less opacification than Group B (p<0.05). Compared with Group B, the mean activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, levels of GSH, and total sulfhydryl contents were higher, whereas the level of malondialdehyde was lower following treatment with hydrogen saline(p<0.05).
This is an initial report showing that hydrogen saline can prevent selenite-induced cataract in rats. It acts via maintaining antioxidant enzymes and GSH, protecting the sulfhydryl group, and inhibiting lipid peroxidation.
PMCID: PMC3731457  PMID: 23922487
11.  Avicin D, a Plant Triterpenoid, Induces Cell Apoptosis by Recruitment of Fas and Downstream Signaling Molecules into Lipid Rafts 
PLoS ONE  2009;4(12):e8532.
Avicins, a family of triterpene electrophiles originally identified as potent inhibitors of tumor cell growth, have been shown to be pleiotropic compounds that also possess antioxidant, anti-mutagenic, and anti-inflammatory activities. We previously showed that Jurkat cells, which express a high level of Fas, are very sensitive to treatment with avicins. Thus, we hypothesized that avicins may induce cell apoptosis by activation of the Fas pathway. By using a series of cell lines deficient in cell death receptors, we demonstrated that upon avicin D treatment, Fas translocates to the cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. In the lipid rafts, Fas interacts with Fas-associated death domain (FADD) and Caspase-8 to form death-inducing signaling complex (DISC) and thus mediates cell apoptosis. Interfering with lipid raft organization by using a cholesterol-depleting compound, methyl-β-cyclodextrin, not only prevents the clustering of Fas and its DISC complex but also reduces the sensitivity of the cells to avicin D. Avicin D activates Fas pathways independent of the association between extracellular Fas ligands and Fas receptors. A deficiency in Fas and its downstream signaling molecules leads to the resistance of the cells to avicin D treatment. Taken together, our results demonstrate that avicin D triggers the redistribution of Fas in the membrane lipid rafts, where Fas activates receptor-mediated cell death.
PMCID: PMC2797328  PMID: 20046832
12.  The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously 
Development (Cambridge, England)  2007;135(1):43-52.
Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.
PMCID: PMC2277323  PMID: 18045837
Uba1; E1; Ubiquitin-activating enzyme; Apoptosis; Proliferation; Drosophila; Autonomous control; Non autonomous control

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