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1.  Chlorella Induces Stomatal Closure via NADPH Oxidase-Dependent ROS Production and Its Effects on Instantaneous Water Use Efficiency in Vicia faba 
PLoS ONE  2014;9(3):e93290.
Reactive oxygen species (ROS) have been established to participate in stomatal closure induced by live microbes and microbe-associated molecular patterns (MAMPs). Chlorella as a beneficial microorganism can be expected to trigger stomatal closure via ROS production. Here, we reported that Chlorella induced stomatal closure in a dose-and time-dependent manner in epidermal peels of Vicia faba. Using pharmacological methods in this work, we found that the Chlorella-induced stomatal closure was almost completely abolished by a hydrogen peroxide (H2O2) scavenger, catalase (CAT), significantly suppressed by an NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), and slightly affected by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), suggesting that ROS production involved in Chlorella-induced stomatal closure is mainly mediated by DPI-sensitive NADPH oxidase. Additionally, Exogenous application of optimal concentrations of Chlorella suspension improved instantaneous water use efficiency (WUEi) in Vicia faba via a reduction in leaf transpiration rate (E) without a parallel reduction in net photosynthetic rate (Pn) assessed by gas-exchange measurements. The chlorophyll fluorescence and content analysis further demonstrated that short-term use of Chlorella did not influence plant photosynthetic reactions center. These results preliminarily reveal that Chlorella can trigger stomatal closure via NADPH oxidase-dependent ROS production in epidermal strips and improve WUEi in leave levels.
PMCID: PMC3970962  PMID: 24687099
2.  Identification of Genes Differentially Expressed between Resistant and Susceptible Tomato Lines during Time-Course Interactions with Xanthomonas perforans Race T3 
PLoS ONE  2014;9(3):e93476.
Bacterial spot caused by several Xanthomonas sp. is one of the most devastating diseases in tomato (Solanum lycopersicum L.). The genetics of hypersensitive resistance to X. perforans race T3 has been intensively investigated and regulatory genes during the infection of race T3 have been identified through transcriptional profiling. However, no work on isolating regulatory genes for field resistance has been reported. In this study, cDNA-amplified fragment length polymorphism technique was used to identify differentially expressed transcripts between resistant tomato accession PI 114490 and susceptible variety OH 88119 at 3, 4 and 5 days post-inoculation of the pathogen. Using 256 selective primer combinations, a total of 79 differentially expressed transcript-derived fragments (TDFs) representing 71 genes were obtained. Of which, 60 were up-regulated and 4 were down-regulated in both tomato lines, 4 were uniquely up-regulated and 2 were uniquely down-regulated in PI 114490, and 1 was specifically up-regulated in OH 88119. The expression patterns of 19 representative TDFs were further confirmed by semi-quantitative and/or quantitative real time RT-PCR. These results suggested that the two tomato lines activated partly similar defensive mechanism in response to race T3 infection. The data obtained here will provide some fundamental information for elucidating the molecular mechanism of response to race T3 infection in tomato plants with field resistance.
PMCID: PMC3970963  PMID: 24686403
3.  PGC1α Expression Defines a Subset of Human Melanoma Tumors with Increased Mitochondrial Capacity and Resistance to Oxidative Stress 
Cancer cell  2013;23(3):287-301.
Cancer cells reprogram their metabolism using different strategies to meet energy and anabolic demands to maintain growth and survival. Understanding the molecular and genetic determinants of these metabolic programs is critical to successfully exploit them for therapy. Here, we report that the oncogenic melanocyte lineage-specification transcription factor MITF drives PGC1α (PPARGC1A) overexpression in a subset of human melanomas and derived cell lines. Functionally, PGC1α positive melanoma cells exhibit increased mitochondrial energy metabolism and ROS detoxification capacities that enables survival under oxidative stress conditions. Conversely, PGC1α negative melanoma cells are more glycolytic and sensitive to ROS-inducing drugs. These results demonstrate that differences in PGC1α levels in melanoma tumors have a profound impact in their metabolism, biology and drug sensitivity.
PMCID: PMC3708305  PMID: 23416000
4.  Different Biosynthesis Patterns among Flavonoid 3-glycosides with Distinct Effects on Accumulation of Other Flavonoid Metabolites in Pears (Pyrus bretschneideri Rehd.) 
PLoS ONE  2014;9(3):e91945.
Flavonoid biosynthesis profile was clarified by fruit bagging and re-exposure treatments in the green Chinese pear ‘Zaosu’ (Pyrus bretschneideri Rehd.) and its red mutant ‘Red Zaosu’. Two distinct biosynthesis patterns of flavonoid 3-glycosides were found in ‘Zaosu’ pear. By comparison with ‘Red Zaosu’, the biosynthesis of flavonoid 3-galactosides and flavonoid 3-arabinosides were inhibited by bagging and these compounds only re-accumulated to a small degree in the fruit peel of ‘Zaosu’ after the bags were removed. In contrast, the biosynthesis of flavonoid 3-gluctosides and flavonoid 3-rutinosides was reduced by bagging and then increased when the fruits were re-exposed to sunlight. A combination of correlation, multicollinearity test and partial-correlation analyses among major flavonoid metabolites indicated that biosynthesis of each phenolic compound was independent in ‘Zaosu’ pear, except for the positive correlation between flavonoid 3-rutincosides and flavanols. In contrast with the green pear cultivar, almost all phenolic compounds in the red mutant had similar biosynthesis patterns except for arbutin. However, only the biosynthesis of flavonoid 3-galactosides was relatively independent and strongly affected the synthesis of the other phenolic compounds. Therefore, we propose a hypothesis that the strong accumulation of flavonoid 3-galactosides stimulated the biosynthesis of other flavonoid compounds in the red mutant and, therefore, caused systemic variation of flavonoid biosynthesis profiles between ‘Zaosu’ and its red mutant. This hypothesis had been further demonstrated by the enzyme activity of UFGT, and transcript levels of flavonoid biosynthetic genes and been well tested by a stepwise linear regression forecasting model. The gene that encodes flavonoid 3-galacosyltransferase was also identified and isolated from the pear genome.
PMCID: PMC3956819  PMID: 24637788
5.  Para-Aminobenzoic Acid (PABA) Synthase Enhances Thermotolerance of Mushroom Agaricus bisporus 
PLoS ONE  2014;9(3):e91298.
Most mushrooms are thermo-sensitive to temperatures over 23°C, which greatly restricts their agricultural cultivation. Understanding mushroom’s innate heat-tolerance mechanisms may facilitate genetic improvements of their thermotolerance. Agaricus bisporus strain 02 is a relatively thermotolerant mushroom strain, while strain 8213 is quite thermo-sensitive. Here, we compared their responses at proteomic level to heat treatment at 33°C. We identified 73 proteins that are differentially expressed between 02 and 8213 or induced upon heat stress in strain 02 itself, 48 of which with a known identity. Among them, 4 proteins are constitutively more highly expressed in 02 than 8213; and they can be further upregulated in response to heat stress in 02, but not in 8213. One protein is encoded by the para-aminobenzoic acid (PABA) synthase gene Pabs, which has been shown to scavenge the reactive oxygen species in vitro. Pabs mRNA and its chemical product PABA show similar heat stress induction pattern as PABA synthase protein and are more abundant in 02, indicating transcriptional level upregulation of Pabs upon heat stress. A specific inhibitor of PABA synthesis impaired thermotolerance of 02, while exogenous PABA or transgenic overexpression of 02 derived PABA synthase enhanced thermotolerance of 8213. Furthermore, compared to 8213, 02 accumulated less H2O2 but more defense-related proteins (e.g., HSPs and Chitinase) under heat stress. Together, these results demonstrate a role of PABA in enhancing mushroom thermotolerance by removing H2O2 and elevating defense-related proteins.
PMCID: PMC3948851  PMID: 24614118
6.  Identification of Metabolic QTLs and Candidate Genes for Glucosinolate Synthesis in Brassica oleracea Leaves, Seeds and Flower Buds 
PLoS ONE  2014;9(3):e91428.
Glucosinolates are major secondary metabolites found in the Brassicaceae family. These compounds play an essential role in plant defense against biotic and abiotic stresses, but more interestingly they have beneficial effects on human health. We performed a genetic analysis in order to identify the genome regions regulating glucosinolates biosynthesis in a DH mapping population of Brassica oleracea. In order to obtain a general overview of regulation in the whole plant, analyses were performed in the three major organs where glucosinolates are synthesized (leaves, seeds and flower buds). Eighty two significant QTLs were detected, which explained a broad range of variability in terms of individual and total glucosinolate (GSL) content. A meta-analysis rendered eighteen consensus QTLs. Thirteen of them regulated more than one glucosinolate and its content. In spite of the considerable variability of glucosinolate content and profiles across the organ, some of these consensus QTLs were identified in more than one tissue. Consensus QTLs control the GSL content by interacting epistatically in complex networks. Based on in silico analysis within the B. oleracea genome along with synteny with Arabidopsis, we propose seven major candidate loci that regulate GSL biosynthesis in the Brassicaceae family. Three of these loci control the content of aliphatic GSL and four of them control the content of indolic glucosinolates. GSL-ALK plays a central role in determining aliphatic GSL variation directly and by interacting epistatically with other loci, thus suggesting its regulatory effect.
PMCID: PMC3948865  PMID: 24614913
7.  Epidemiological and Clinical Characteristics and Risk Factors for Death of Patients with Avian Influenza A H7N9 Virus Infection from Jiangsu Province, Eastern China 
PLoS ONE  2014;9(3):e89581.
A novel avian influenza A (H7N9) virus has caused great morbidity as well as mortality since its emergence in Eastern China in February 2013. However, the possible risk factors for death are not yet fully known.
Methods and Findings
Patients with H7N9 virus infection between March 1 and August 14, 2013 in Jiangsu province were enrolled. Data were collected with a standard form. Mean or percentage was used to describe the features, and Fisher's exact test or t-test test was used to compare the differences between fatal and nonfatal cases with H7N9 virus infection. A total of 28 patients with H7N9 virus infection were identified among whom, nine (32.1%) died. The median age of fatal cases was significant higher than nonfatal cases (P<0.05). Patients with older age were more strongly associated with increased odds of death (OR = 30.0; 95% CI, 2.85–315.62). Co-morbidity with chronic lung disease and hypertension were risk factors for mortality (OR = 14.40; 95% CI, 1.30–159.52, OR = 6.67; 95% CI, 1.09–40.43, respectively). Moreover, the presence of either bilateral lung inflammation or pulmonary consolidation on chest imaging on admission was related with fatal outcome (OR = 7.00; 95%CI, 1.10–44.61). Finally, dynamic monitoring showed that lymphopenia was more significant in fatal group than in nonfatal group from day 11 to week five (P<0.05). The decrease in oxygenation indexes were observed in most cases and more significantly in fatal cases after week three (P<0.05), and the value of nearly all fatal cases were below 200 mmHg during our evaluation period.
Among cases with H7N9 virus infection, increased age accompanied by co-morbidities was the risk of death. The severity of lung infection at admission, the persistence of lymphocytopenia, and the extended duration of lower oxygenation index all contributed to worsened outcomes of patients with H7N9 virus infection.
PMCID: PMC3942409  PMID: 24595034
8.  A Comparison of Screening Methods to Identify Waterlogging Tolerance in the Field in Brassica napus L. during Plant Ontogeny 
PLoS ONE  2014;9(3):e89731.
Waterlogging tolerance is typically evaluated at a specific development stage, with an implicit assumption that differences in waterlogging tolerance expressed in these systems will result in improved yield performance in fields. It is necessary to examine these criteria in fields.
In the present study, three experiments were conducted to screen waterlogging tolerance in 25 rapeseed (Brassica napus L.) varieties at different developmental stages, such as seedling establishment stage and seedling stage at controlled environment, and maturity stage in the fields. The assessments for physiological parameters at three growth stages suggest that there were difference of waterlogging tolerance at all the development stages, providing an important basis for further development of breeding more tolerant materials. The results indicated that flash waterlogging restricts plant growth and growth is still restored after removal of the stress. Correlation analysis between waterlogging tolerance coefficient (WTC) of yield and other traits revealed that there was consistency in waterlogging tolerance of the genotypes until maturity, and good tolerance at seedling establishment stage and seedling stage can guarantee tolerance in later stages. The waterlogging-tolerant plants could be selected using some specific traits at any stage, and selections would be more effective at the seedling establishment stage.
Thus, our study provides a method for screening waterlogging tolerance, which would enable the suitable basis for initial selection of a large number of germplasm or breeding populations for waterlogging tolerance and help for verifying their potential utility in crop-improvement.
PMCID: PMC3940661  PMID: 24594687
9.  FOXP3 hypermethylation is associated with diesel exhaust exposure and risk for childhood asthma 
PMCID: PMC3563724  PMID: 23260754
DNA methylation; respiratory hypersensitivity; saliva; pyrosequencing; wheezing; traffic-related air pollution
10.  Managing Potato Biodiversity to Cope with Frost Risk in the High Andes: A Modeling Perspective 
PLoS ONE  2014;9(1):e81510.
Austral summer frosts in the Andean highlands are ubiquitous throughout the crop cycle, causing yield losses. In spite of the existing warming trend, climate change models forecast high variability, including freezing temperatures. As the potato center of origin, the region has a rich biodiversity which includes a set of frost resistant genotypes. Four contrasting potato genotypes –representing genetic variability- were considered in the present study: two species of frost resistant native potatoes (the bitter Solanum juzepczukii, var. Luki, and the non-bitter Solanum ajanhuiri, var. Ajanhuiri) and two commercial frost susceptible genotypes (Solanum tuberosum ssp. tuberosum var. Alpha and Solanum tuberosum ssp. andigenum var. Gendarme). The objective of the study was to conduct a comparative growth analysis of four genotypes and modeling their agronomic response under frost events. It included assessing their performance under Andean contrasting agroecological conditions. Independent subsets of data from four field experiments were used to parameterize, calibrate and validate a potato growth model. The validated model was used to ascertain the importance of biodiversity, represented by the four genotypes tested, as constituents of germplasm mixtures in single plots used by local farmers, a coping strategy in the face of climate variability. Also scenarios with a frost routine incorporated in the model were constructed. Luki and Ajanhuiri were the most frost resistant varieties whereas Alpha was the most susceptible. Luki and Ajanhuiri, as monoculture, outperformed the yield obtained with the mixtures under severe frosts. These results highlight the role played by local frost tolerant varieties, and featured the management importance –e.g. clean seed, strategic watering- to attain the yields reported in our experiments. The mixtures of local and introduced potatoes can thus not only provide the products demanded by the markets but also reduce the impact of frosts and thus the vulnerability of the system to abiotic stressors.
PMCID: PMC3907385  PMID: 24497912
11.  Plakophilin-3 Catenin Associates with the ETV1/ER81 Transcription Factor to Positively Modulate Gene Activity 
PLoS ONE  2014;9(1):e86784.
Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm (“animal cap”) cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.
PMCID: PMC3903613  PMID: 24475179
12.  Genetic Characteristics of the Coxsackievirus A24 Variant Causing Outbreaks of Acute Hemorrhagic Conjunctivitis in Jiangsu, China, 2010 
PLoS ONE  2014;9(1):e86883.
During September 2010, an outbreak of acute hemorrhagic conjunctivitis reemerged in Jiangsu, three years after the nationwide epidemic in China in 2007. In total, 2409 cases were reported, 2118 of which were reported in September; 79.8% of those affected were students or teachers, with a median age of 16 years. To identify and demonstrate the genetic characteristics of the etiological agent, 52 conjunctival swabs were randomly collected from four different cities. After detection and isolation, 43 patients were positive for coxsackievirus A24 variant according to PCR and 20 according to culture isolation. Neither adenovirus nor EV70 was detected. A phylogenetic study of the complete 3Cpro and VP1 regions showed that the Jiangsu isolates clustered into a new lineage, GIV-C5, with two uniform amino-acid mutations that distinguished them from all previous strains. Another new cluster, GIV-C4, formed by Indian isolates from 2007 and Brazilian isolates from 2009, was also identified in this study. Interestingly, our isolates shared greatest homology with the GIV-C4 strains, not with the isolates that were responsible for the nationwide acute hemorrhagic conjunctivitis epidemic in China in 2007. Although all our isolates were closely related, they could be differentiated into two subclusters within GIV-C5. In conclusion, our study suggests that a new cluster of coxsackievirus A24 variant that had already evolved into diverse strains was associated with the acute hemorrhagic conjunctivitis outbreaks in Jiangsu in September 2010. These viruses might have originated from the virus isolated in India in 2007, rather than from the epidemic strains isolated in China in 2007.
PMCID: PMC3901726  PMID: 24475191
13.  LcMYB1 Is a Key Determinant of Differential Anthocyanin Accumulation among Genotypes, Tissues, Developmental Phases and ABA and Light Stimuli in Litchi chinensis 
PLoS ONE  2014;9(1):e86293.
The red coloration of litchi fruit depends on the accumulation of anthocyanins. The anthocyanins level in litchi fruit varies widely among cultivars, developmental stages and environmental stimuli. Previous studies on various plant species demonstrate that anthocyanin biosynthesis is controlled at the transcriptional level. Here, we describe a litchi R2R3-MYB transcription factor gene, LcMYB1, which demonstrates a similar sequence as other known anthocyanin regulators. The transcription levels of the LcMYB1 and anthocyanin biosynthetic genes were investigated in samples with different anthocyanin levels. The expression of LcMYB1 was strongly associated with tissue anthocyanin content. LcMYB1 transcripts were only detected in anthocyanin-accumulating tissues and were positively correlated with anthocyanin accumulation in the pericarps of 12 genotypes. ABA and sunlight exposure promoted, whereas CPPU and bagging inhibited the expression of LcMYB1 and anthocyanin accumulation in the pericarp. Cis-elements associated with light responsiveness and abscisic acid responsiveness were identified in the promoter region of LcMYB1. Among the 6 structural genes tested, only LcUFGT was highly correlated with LcMYB1. These results suggest that LcMYB1 controls anthocyanin biosynthesis in litchi and LcUFGT might be the structural gene that is targeted and regulated by LcMYB1. Furthermore, the overexpression of LcMYB1 induced anthocyanin accumulation in all tissues in tobacco, confirming the function of LcMYB1 in the regulation of anthocyanin biosynthesis. The upregulation of NtAn1b in response to LcMYB1 overexpression seems to be essential for anthocyanin accumulation in the leaf and pedicel. In the reproductive tissues of transgenic tobacco, however, increased anthocyanin accumulation is independent of tobacco's endogenous MYB and bHLH transcriptional factors, but associated with the upregulation of specific structural genes.
PMCID: PMC3897698  PMID: 24466010
14.  The Phloem-Sap Feeding Mealybug (Ferrisia virgata) Carries ‘Candidatus Liberibacter asiaticus’ Populations That Do Not Cause Disease in Host Plants 
PLoS ONE  2014;9(1):e85503.
‘Candidatus Liberibacter asiaticus’ (Las) is the primary causal agent of huanglongbing (HLB), the most devastating disease of citrus worldwide. There are three known insect vectors of the HLB-associated bacteria, and all are members of the Hemiptera: Diaphorina citri (Psyllidae), Trioza erytreae (Triozidae), and Cacopsylla (Psylla) citrisuga (Psyllidae). In this study, we found that another hemipteran, the striped mealybug Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae), was able to acquire and retain Las bacteria. The bacterial titers were positively correlated with the feeding acquisition time on Las-infected leaf discs, with a two-weeks feeding period resulting in Ct values ranging from 23.1 to 36.1 (8.24×107 to 1.07×104 Las cells per mealybug). We further discovered that the prophage/phage populations of Las in the mealybugs were different from those of Las in psyllids based on Las prophage-specific molecular markers: infected psyllids harbored the Las populations with prophage/phage FP1 and FP2, while infected mealybugs carried the Las populations with the iFP3 being the dominant prophage/phage. As in the psyllids, Las bacteria were shown to move through the insect gut wall to the salivary glands after being ingested by the mealybug based on a time-course quantitative polymerase chain reaction (qPCR) assay of the dissected digestive systems. However, Las populations transmitted by the mealybugs did not cause disease in host plants. This is the first evidence of genetic difference among Las populations harbored by different insect vectors and difference among Las populations with respect to whether or not they cause disease in host plants.
PMCID: PMC3896372  PMID: 24465578
15.  Hydrogen Sulfide Prolongs Postharvest Storage of Fresh-Cut Pears (Pyrus pyrifolia) by Alleviation of Oxidative Damage and Inhibition of Fungal Growth 
PLoS ONE  2014;9(1):e85524.
Hydrogen sulfide (H2S) has proved to be a multifunctional signaling molecule in plants and animals. Here, we investigated the role of H2S in the decay of fresh-cut pears (Pyrus pyrifolia). H2S gas released by sodium hydrosulfide (NaHS) prolonged the shelf life of fresh-cut pear slices in a dose-dependent manner. Moreover, H2S maintained higher levels of reducing sugar and soluble protein in pear slices. H2S significantly reduced the accumulation of hydrogen peroxide (H2O2), superoxide radicals (•O2−) and malondialdehyde (MDA). Further investigation showed that H2S fumigation up-regulated the activities of antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT), and guaiacol peroxidase (POD), while it down-regulated those of lipoxygenase (LOX), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO). Furthermore, H2S fumigation effectively inhibited the growth of two fungal pathogens of pear, Aspergillus niger and Penicillium expansum, suggesting that H2S can be developed as an effective fungicide for postharvest storage. The present study implies that H2S is involved in prolonging postharvest storage of pears by acting as an antioxidant and fungicide.
PMCID: PMC3893216  PMID: 24454881
16.  Isolation of a Novel Peroxisomal Catalase Gene from Sugarcane, Which Is Responsive to Biotic and Abiotic Stresses 
PLoS ONE  2014;9(1):e84426.
Catalase is an iron porphyrin enzyme, which serves as an efficient scavenger of reactive oxygen species (ROS) to avoid oxidative damage. In sugarcane, the enzymatic activity of catalase in a variety (Yacheng05–179) resistant to the smut pathogen Sporisorium scitamineum was always higher than that of the susceptible variety (Liucheng03–182), suggesting that catalase activity may have a positive correlation with smut resistance in sugarcane. To understand the function of catalase at the molecular level, a cDNA sequence of ScCAT1 (GenBank Accession No. KF664183), was isolated from sugarcane infected by S. scitamineum. ScCAT1 was predicted to encode 492 amino acid residues, and its deduced amino acid sequence shared a high degree of homology with other plant catalases. Enhanced growth of ScCAT1 in recombinant Escherichia coli Rosetta cells under the stresses of CuCl2, CdCl2 and NaCl indicated its high tolerance. Q-PCR results showed that ScCAT1 was expressed at relatively high levels in the bud, whereas expression was moderate in stem epidermis and stem pith. Different kinds of stresses, including S. scitamineum challenge, plant hormones (SA, MeJA and ABA) treatments, oxidative (H2O2) stress, heavy metal (CuCl2) and hyper-osmotic (PEG and NaCl) stresses, triggered a significant induction of ScCAT1. The ScCAT1 protein appeared to localize in plasma membrane and cytoplasm. Furthermore, histochemical assays using DAB and trypan blue staining, as well as conductivity measurement, indicated that ScCAT1 may confer the sugarcane immunity. In conclusion, the positive response of ScCAT1 to biotic and abiotic stresses suggests that ScCAT1 is involved in protection of sugarcane against reactive oxidant-related environmental stimuli.
PMCID: PMC3879312  PMID: 24392135
17.  Plant Growth Retardation and Conserved miRNAs Are Correlated to Hibiscus Chlorotic Ringspot Virus Infection  
PLoS ONE  2013;8(12):e85476.
Virus infection may cause a multiplicity of symptoms in their host including discoloration, distortion and growth retardation. Hibiscus chlorotic ringspot virus (HCRSV) infection was studied using kenaf (Hibiscus cannabinus L.), a non-wood fiber-producing crop in this study. Infection by HCRSV reduced the fiber yield and concomitant economic value of kenaf. We investigated kenaf growth retardation and fluctuations of four selected miRNAs after HCRSV infection. Vegetative growth (including plant height, leaf size and root development) was severely retarded. From the transverse and radial sections of the mock and HCRSV-infected kenaf stem, the vascular bundles of HCRSV-infected plants were severely disrupted. In addition, four conserved plant developmental and defence related microRNAs (miRNAs) (miR165, miR167, miR168 and miR171) and their respective target genes phabulosa (PHB), auxin response factor 8 (ARF8), argonaute 1 (AGO1) and scarecrow-like protein 1 (SCL1) displayed variation in expression levels after HCRSV infection. Compared with the mock inoculated kenaf plants, miR171 and miR168 and their targets SCL1 and AGO1 showed greater fluctuations after HCRSV infection. As HCRSV upregulates plant SO transcript in kenaf and upregulated AGO1 in HCRSV-infected plants, the expression level of AGO1 transcript was further investigated under sulfite oxidase (SO) overexpression or silencing condition. Interestingly, the four selected miRNAs were also up- or down-regulated upon overexpression or silencing of SO. Plant growth retardation and fluctuation of four conserved miRNAs are correlated to HCRSV infection.
PMCID: PMC3875576  PMID: 24386476
18.  Single-Walled Carbon Nanotubes Alter Cytochrome C Electron Transfer and Modulate Mitochondrial Function 
ACS nano  2012;6(12):10486-10496.
Single-walled carbon nanotubes (SWCNTs) are broadly used for various biomedical applications such as drug delivery, in vivo imaging and cancer photothermal therapy due to their unique physiochemical properties. However, once they enter the cells, the effects of SWCNTs to the intracellular organelles and macromolecules are not comprehensively understood. Cytochrome c (Cyt c), as a key component of the electron transport chain in mitochondria, plays an essential role in cellular energy consumption, growth and differentiation. In this study, we found the mitochondrial membrane potential (MMP) and mitochondrial oxygen uptake were greatly decreased in human epithelial KB cells treated with SWCNTs, which accompanies the reduction of Cyt c. SWCNTs deoxidized Cyt c in a pH dependent manner as evidenced by the appearance of a 550 nm characteristic absorption peak, which intensity increased as pH increase. Circular dichroism measurement confirmed the pH-dependent conformational change, which facilitated closer association of SWCNTs with the heme pocket of Cyt c and thus expedited the reduction of Cyt c. The electron transfer of Cyt c is also disturbed by SWCNTs, as measured with electron spin resonance spectroscopy. In conclusion, the redox activity of Cyt c was affected by SWCNTs treatment due to attenuated electron transfer and conformational change of Cyt c, which consequently changed mitochondrial respiration of SWCNTs treated cells. This work is significant to SWCNTs research because it provided novel understanding to the disruption of SWCNTs to the mitochondria and has important implications for biomedical applications of SWCNTs.
PMCID: PMC3548237  PMID: 23171082
Single-walled carbon nanotubes; Carboxy; Cytochrome C; Electron transfer; Mitochondrial function; Redox activity
19.  Characterization of a Pathogen Induced Thaumatin-Like Protein Gene AdTLP from Arachis diogoi, a Wild Peanut 
PLoS ONE  2013;8(12):e83963.
Peanut (Arachis hypogaea L) is one of the widely cultivated and leading oilseed crops of the world and its yields are greatly affected by various biotic and abiotic stresses. Arachis diogoi, a wild relative of peanut, is an important source of genes for resistance against various stresses that affect peanut. In our previous study a thaumatin-like protein gene was found to be upregulated in a differential expression reverse transcription PCR (DDRT-PCR) study using the conidial spray of the late leaf spot pathogen, Phaeoisariopsis personata. In the present study, the corresponding full length cDNA was cloned using RACE-PCR and has been designated as AdTLP. It carried an open reading frame of 726 bp potentially capable of encoding a polypeptide of 241 amino acids with 16 conserved cysteine residues. The semi-quantitative RT-PCR analysis showed that the transcript level of AdTLP increased upon treatment with the late leaf spot pathogen of peanut, P. personata and various hormone treatments indicating its involvement in both, biotic and abiotic stresses. The antifungal activity of the purified recombinant protein was checked against different fungal pathogens, which showed enhanced anti-fungal activity compared to many other reported TLP proteins. The recombinant AdTLP-GFP fusion protein was found to be predominantly localized to extracellular spaces. Transgenic tobacco plants ectopically expressing AdTLP showed enhanced resistance to fungal pathogen, Rhizoctonia solani. The seedling assays showed enhanced tolerance of AdTLP transgenic plants against salt and oxidative stress. The transcript analysis of various defense related genes highlighted constitutively higher level expression of PR1a, PI-I and PI-II genes in transgenic plants. These results suggest that the AdTLP is a good candidate gene for enhancing stress resistance in crop plants.
PMCID: PMC3868660  PMID: 24367621
20.  Increased expression of chymase in inflammatory polyps in elderly patients with functional bowel disorder 
Chymase, a chymotrypsin-like protease, is a non-angiotensin-converting enzyme (ACE) angiotensin II (Ang II)-generating enzyme. The aim of the present study was to investigate whether chymase activity was increased in inflammatory polyps of elderly patients with functional bowel disorder (FBD). This study enrolled 45 elderly patients with FBD and 44 healthy control individuals. Expression of chymase in intestinal mucosa was assessed using fluorescence quantitative polymerase chain reaction and immunohistochemistry (IHC). IHC showed an increased number of chymase-positive mast cells in inflammatory polyps than in healthy intestinal mucosa (P<0.05). Compared with healthy mucosa, expression of chymase at the mRNA and protein level was significantly higher in inflammatory polyps. The frequencies of the chymase GG genotype and the G allele type were higher in the intestinal mucosa of patients with FBD compared with healthy controls (66.67 versus 40.91%, 81.11 versus 63.63%, both P<0.05). The frequency of the G allele type in the intestinal mucosa of the C4 subgroup of FBD was higher than that in the control group. However, in other FBD subgroups, there was no difference between patients and controls. Based on the fact that enhanced chymase expression was observed in inflammatory polyps of elderly patients with FBD relative to those in healthy controls, it was concluded that chymase has a significant role in the pathogenesis of inflammatory polyps in elderly patients with FBD.
PMCID: PMC3881056  PMID: 24396407
chymase; functional bowel disorder; elderly; intestinal inflammatory polyps
21.  Unmarried male migrants and sexual risk behavior: a cross-sectional study in Shanghai, China 
BMC Public Health  2013;13:1152.
In China, there is increasing concern because of the rapid increase in HIV infection recorded over recent years. Migrant workers are recognized as one of the groups most affected. In this study, HIV/AIDS-related knowledge, attitudes, and behavior among unmarried migrant workers in Shanghai are investigated, with the aim of providing critical information for policy makers and sex educators to reinforce sexual health services and sex health education targeting the behavior and sexual health of unmarried male migrants.
A cross-sectional survey was conducted among unmarried male migrant workers in Shanghai, China’s largest city and housing the most migrants. A self-administered, anonymous questionnaire was used to collect information on knowledge, attitudes, and behavior associated with increased risk of HIV/AIDS.
A total of 2254 subjects were questioned, with a response rate of 91.3%. Among those interviewed, 63.5% reported sexual activities. Misconceptions regarding HIV transmission, poor perception of HIV infection, and low use of condoms were not uncommon. Among those who had sexual intercourse, 73.7% had not used condoms in their last sexual intercourse, and 28.6% reported having engaged in sexual risk behavior (defined as having at least one non-regular partner). Multivariate logistic regression analyses identified several indicators of sexual risk behavior, including younger age at first sexual intercourse (OR: 0.67, 95% CI: 0.31–0.91 for older age at first sexual intercourse), more cities of migration (OR: 2.91, 95% CI: 2.17–3.81 for high level; OR: 1.15, 95% CI: 1.06–1.29 for medium level), poor perception of acquiring HIV/AIDS (OR: 1.52, 95% CI: 1.33–1.96 for unlikely; OR: 2.38, 95% CI: 1.61–3.70 for impossible), frequent exposure to pornography (OR: 0.33, 95% CI: 0.11–0.43 for never; OR: 0.69, 95% CI: 0.60–1.81 for less frequently), not knowing someone who had or had died of HIV/AIDS and related diseases (OR: 2.13, 95% CI: 1.70–2.53 for no), and having peers who engaged in sex with a non-regular sex partner (OR: 4.40, 95% CI: 3.37–5.56 for yes).
Today, it is necessary to reinforce sex health education among unmarried migrants and sexual health services should target vulnerable migrant young people.
PMCID: PMC3903055  PMID: 24321180
22.  Identification of novel splice variants and exons of human endothelial cell-specific chemotaxic regulator (ECSCR) by bioinformatics analysis 
Recent discovery of biological function of endothelial cell-specific chemotaxic regulator (ECSCR), previously known as endothelial cell-specific molecule 2 (ECSM2), in modulating endothelial cell migration, apoptosis, and angiogenesis, has made it an attractive molecule in vascular research. Thus, identification of splice variants of ECSCR could provide new strategies for better understanding its roles in health and disease. In this study, we performed a series of blast searches on the human EST database with known ECSCR cDNA sequence (Variant 1), and identified additional three splice variants (Variants 2–4). When examining the ECSCR gene in the human genome assemblies, we found a large unknown region between Exons 9 and 11. By PCR amplification and sequencing, we partially mapped Exon 10 within this previously unknown region of the ECSCR gene. Taken together, in addition to previously reported human ECSCR, we identified three novel full-length splice variants potentially encoding different protein isoforms. We further defined a total of twelve exons and nearly all exon-intron boundaries of the gene, of which only eight are annotated in current public databases. Our work provides new information on gene structure and alternative splicing of the human ECSCR, which may imply its functional complexity. This undoubtedly opens new opportunities for future investigation of the biological and pathological significance of these ECSCR splice variants.
PMCID: PMC3513626  PMID: 23147565
ECSCR (ECSM2); alternative splicing; data mining; EST alignment; sequence homology; exon-intron boundary
23.  Botulinum Toxin in the Treatment of Drooling in Tetraplegic Patients With Brain Injury 
Annals of Rehabilitation Medicine  2013;37(6):796-803.
To investigate the effect of botulinum toxin type A (BTA) injection into the salivary gland and to evaluate the changes of drooling in varied postures in tetraplegic patients with brain injury.
Eight tetraplegic patients with brain injury were enrolled. BTA was injected into each parotid and submandibular gland of both sides under ultrasonographic guidance. Drooling was measured by a questionnaire-based scoring system for drooling severity and frequency, and the sialorrhea was measured by a modified Schirmer test for the patients before the injection, 3 weeks and 3 months after the injection. Drooling was evaluated in each posture, such as supine, sitting, and tilt table standing, and during involuntary mastication, before and after the injection.
The severity and frequency of drooling and the modified Schirmer test improved significantly at 3 weeks and 3 months after the injection (p<0.05). Drooling was more severe and frequent in tilt table standing than in the sitting position and in sitting versus supine position (p<0.05). The severity of drooling was significantly increased in the patients with involuntary mastication (p<0.05).
Salivary gland injection of BTA in patients with tetraplegia resulting from brain injury who had drooling and sialorrhea could improve the symptoms for 3 months without complications. The severity and frequency of drooling were dependent on posture and involuntary mastication. Proper posture and involuntary mastication of the patients should be taken into account in planning drooling treatment.
PMCID: PMC3895519  PMID: 24466514
Botulinum toxins; Drooling; Posture; Salivary glands; Ultrasonography
24.  The Invasive Wetland Plant Alternanthera philoxeroides Shows a Higher Tolerance to Waterlogging than Its Native Congener Alternanthera sessilis 
PLoS ONE  2013;8(11):e81456.
Plant invasion is one of the major threats to natural ecosystems. Phenotypic plasticity is considered to be important for promoting plant invasiveness. High tolerance of stress can also increase survival of invasive plants in adverse habitats. Limited growth and conservation of carbohydrate are considered to increase tolerance of flooding in plants. However, few studies have examined whether invasive species shows a higher phenotypic plasticity in response to waterlogging or a higher tolerance of waterlogging (lower plasticity) than native species. We conducted a greenhouse experiment to compare the growth and morphological and physiological responses to waterlogging of the invasive, clonal, wetland species Alternanthera philoxeroides with those of its co-occurring, native, congeneric, clonal species Alternanthera sessilis. Plants of A. philoxeroides and A. sessilis were subjected to three treatments (control, 0 and 60 cm waterlogging). Both A. philoxeroides and A. sessilis survived all treatments. Overall growth was lower in A. philoxeroides than in A. sessilis, but waterlogging negatively affected the growth of A. philoxeroides less strongly than that of A. sessilis. Alternanthera philoxeroides thus showed less sensitivity of growth traits (lower plasticity) and higher waterlogging tolerance. Moreover, the photosynthetic capacity of A. philoxeroides was higher than that of A. sessilis during waterlogging. Alternanthera philoxeroides also had higher total non-structural and non-soluble carbohydrate concentrations than A. sessilis at the end of treatments. Our results suggest that higher tolerance to waterlogging and higher photosynthetic capacity may partly explain the invasion success of A. philoxeroides in wetlands.
PMCID: PMC3841148  PMID: 24303048
25.  Effects of recombinant human interleukin-10 on Treg cells, IL-10 and TGF-β in transplantation of rabbit skin 
Molecular Medicine Reports  2013;9(2):639-644.
The current study aimed to investigate the rejection and survival time of grafted skin, and the changes of Treg cells, interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) in peripheral blood following skin transplantation with recombinant human interleukin-10 (rhIL-10) or cyclosporin A (CsA), as well as the role of IL-10 in immunological rejection mechanisms. A total of 36 rabbits were divided into two groups. The skin of a donor rabbit was transplanted onto the back of one receptor rabbit. Receptors were randomly divided into six groups, including rhIL-10 low-dose (5 μg/kg/d), rhIL-10 high-dose (10 μg/kg/d), CsA low-dose (5 mg/kg/d), CsA high-dose (10 mg/kg/d), rhIL-10 (5 μg/kg/d) and CsA (5 mg/kg/d) and negative control normal saline (NS; 1 ml/d). All groups received intramuscular drug injection for ten days, beginning one day prior to skin transplantation surgery. Following transplantation, each rabbit’s peripheral blood was collected at different times. The changes of CD4+CD25+ regulatory T cells, IL-10 and TGF-β were determined by flow cytometry and enzyme-linked immunosorbent assay. When compared with the control group, the rejection and survival times of the experimental groups were longer following skin graft. Compared with the two CsA groups and the control group, the proportion of CD4+CD25+ regulatory T cells of rhIL-10 groups was significantly upregulated on the 4th and 7th days following surgery. However, TGF-β levels were not significantly different. Data suggested that the concentration of IL-10 was positively correlated with the proportion of CD4+CD25+ regulatory T cells. In addition, IL-10 may delay the rejection time of rabbit skin transplantation and prolong the survival time. Thus, the role of IL-10 in inhibited allograft rejection may be associated with CD4+CD25+ regulatory T cells and IL-10, and may be independent of TGF-β.
PMCID: PMC3896515  PMID: 24270972
cytokines; graft rejection; interleukin-10; Treg cells

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