Expressions of glucose transporter genes are diversely attenuated and significantly associated with prostate cancer progression.

Prostate cancer is a health-threaten disease in men worldwide, however, lacking is the reliable biomarkers for patient management. Aberrant metabolic events including glucose metabolism are involved in prostate cancer progression. To examine the involvement of glucose metabolic pathways in prostate cancer, we analyzed the expression profiles of glucose transporter family genes using multiple RNA-seq datasets. Our results showed that three SLC2A family genes (SLC2A4/5/9) were significantly downregulated in primary prostate cancers compared to their benign compartments. These down-regulated expressions were inversely correlated with their gene promoter methylation and genome abnormalities. Among these three SLC2A genes, only SLC2A4 showed a significantly reverse correlation with all clinicopathological parameters, including TNM stage, disease relapse, Gleason score, disease-specific survival, and progression-free interval. In addition, the expression levels of these three genes were strongly correlated with anti-cancer immune cell filtration in primary prostate cancers. In a group of patients with early-onset prostate cancers, SLC2A4 also showed a strong negative correlation with multiple clinicopathological parameters, such as tumor mutation burden, biochemical relapse, pre-surgical PSA levels, and Gleason score but a positive correlation with progression-free interval after surgery. In metastatic castration-resistant prostate cancers (CRPC), SLC2A9 gene expression but not SLC2A4 or SLC2A5 genes showed a significant correlation with androgen receptor (AR) activity score and neuroendocrinal (NE) activity score. Meanwhile, SLC2A2/9/13 expression was significantly elevated in CRPC tumors with neuroendocrinal features compared to those without NE features. On the other hand, SLC2A10 and SlC2A12 gene expression were significantly reduced in NEPC tumors compared to CRPC tumors. Consistently, SLC2A10/12 expression levels were significantly reduced in castrated animals carrying the LuCaP35 xenograft models. Survival outcome analysis revealed that SLC2A4 expression in primary tumors is a favorable prognostic factor and SLC2A6 is a worse prognostic factor for disease-specific survival and progression-free survival in prostate cancer patients. In conclusion, our results suggest that SLC2A4/6 expressions are strong prognostic factors for prostate cancer progression and survival. The significance of SLC2A2/9/13 over-expression during NEPC progression needs more investigation.

High-throughput transcriptome sequencing reveals the protective role of adenosine receptor-related genes in paraquat-exposed Caenorhabditis elegans.

This study sought to identify the genes associated with adenosine's protective action against paraquat (PQ)-induced oxidative stress via the adenosine receptor (ADOR-1) in Caenorhabditis elegans (C. elegans). The C. elegans was divided into 3 groups-2 groups exposed to PQ, one in presence, and one in absence of adenosine-and a control group that was not treated. Each group's total RNA was extracted and sequenced. When the transcriptomes of these groups were analyzed, several genes were found to be differently expressed. These differentially expressed genes were significantly enriched in adenosine-response biological processes and pathways, including gene ontology terms related to neuropeptide and kyoto encyclopedia of genes and genomes pathways associated to cAMP pathway regulator activity. Quantitative reverse-transcription PCR confirmed that G-protein-coupled receptors signaling pathway involving dop-1, egl-30, unc-13, kin-1, and goa-1 genes may play crucial roles in modulating adenosine's protective action. Interestingly, there are no significant variations in the expression of the ador-1 gene across the 3 treatments, thereby indicating that adenosine receptor exerts a consistent and stable influence on its related pathways irrespective of the presence or absence of PQ. Furthermore, the wild-type group with ador-1 gene has higher survival rate than that of the ador-1-/RNA interference group while treated with PQ in the presence of adenosine. Conclusively, our study uncovered a number of novel PQ-response genes and adenosine receptor-related genes in C. elegans, which may function as major regulators of PQ-induced oxidative stress and indicate the possible protective effects of adenosine.

High-performance colorimetric immunoassay for determination of chloramphenicol using metal-organic framework-based hybrid composites with increased peroxidase activity.

Metal-organic frameworks (MOFs) as carriers for high-capacity loading of HRP-IgG and gold nanoparticles are introduced, to prepare MOF hybrids with enhanced peroxidase activity. The prepared MOF hybrids were employed to establish an indirect competitive colorimetric immunoassay for chloramphenicol (CAP) detection, in which the limit of detection for CAP is 0.006 µg·L-1, only one-fifth of that of the conventional ELISA using the same antibodies and antigens. The linear range was 0.008-0.108 µg·L-1, and the recovery of spiked milk samples varied in the range 76.0-106.0% through three independent experiments. Our proposed colorimetric immunoassay using the MOF hybrid immunoprobe provides a novel platform for ultra-sensitive determination of CAP residues, and it also could be used as a signal amplification model for the high-performance colorimetric immunoassay in food safety monitoring.

Effect of Adenosine Receptor Antagonists on Adenosine-Pretreated PC12 Cells Exposed to Paraquat.

Previous studies evaluated the adenosine receptor antagonists alone to determine their effects on oxidative stress, but little is known about adenosine's protective efficacy when oxidative injury occurs in vivo. Adenosine is a crucial signaling molecule recognized by four distinct G-protein-coupled receptors (GPCRs) (i.e., A1R, A2AR, A2BR, and A3R) and protects cells against pathological conditions. The present study was performed to evaluate the role of antagonist modulation in the setting of paraquat toxicity with adenosine pretreatment. First, PC12 cells were exposed to paraquat (850 µM) and adenosine (30 µM) to develop an in vitro model for the antagonist effect assay. Second, we found that the A1R antagonist DPCPX enhanced the viability of paraquat-induced PC12 cells that underwent adenosine pretreatment. Moreover, the A2AR antagonist ZM241385 decreased the viability of paraquat-induced PC12 cells that underwent adenosine pretreatment. Our findings indicate that adenosine protection requires a dual blockade of A1R and activation of A2AR to work at its full potential, and the A2B and A3 adenosine receptor antagonists increased paraquat-induced oxidative damage. This represents a novel pharmacological strategy based on A1/A2A interactions and can assist in clarifying the role played by AR antagonists in the treatment of neurodegenerative diseases.

AdoR-1 (Adenosine Receptor) Contributes to Protection against Paraquat-Induced Oxidative Stress in Caenorhabditis elegans.

AdoR-1, the single adenosine receptor homolog in Caenorhabditis elegans, which belongs to the superfamily of G-protein coupled receptors (GPCRs), mediates most of the physiological effects of extracellular adenosine. Adenosine has been proved to improve the survival rate of C. elegans in oxidative stress conditions. However, the potential mechanism of adenosine's protective effect against oxidative stress via AdoR-1 has not been studied. In this study, C. elegans were divided into three groups: two groups with paraquat treatment, one in the presence and one in the absence of adenosine, and an untreated control group. Results indicate that many differentially expressed genes were found to be enriched significantly in neural-related signaling pathways among transcriptome data of three groups. Further gene network analysis showed that some important genes well known to be involved in promoting the acetylcholine release pathway, such as dop-1, egl-30, and unc-13, and those involved in promoting the neuropeptide release pathway, such as kin-1, were upregulated by paraquat induction but downregulated after adenosine treatment. Meanwhile, a completely opposite trend was observed for the goa-1 gene that inhibits the acetylcholine-release and neuropeptide-release pathway. Additionally, some biochemical assays including SOD, GSSG, GSH, and AChE were measured to identify the potential protection of adenosine against oxidative stress between wild-type strain N2 and ador-1 gene knockout strain EG6890. Conclusively, our study revealed series of adenosine receptor-mediated genes in C. elegans that might act as regulators of paraquat-induced oxidative stress and may indicate adenosine's promising protective effects.

Optimization of In Vitro Culture Conditions for Production of Cordyceps bassiana Spores (Ascomycetes) and the Effect of Spore Extracts on the Health of Caenorhabditis elegans.

Cordyceps bassiana, an entomogenous fungus, has been used as a dietary therapeutic in traditional Chinese medicine for several millennia. However, naturally occurring C. bassiana is notably scarce in the field, especially at the spore stage. To date, there are few comprehensive studies on the biological characteristics and pharmacological attributes of C. bassiana spores. In this study, an isolate of C. bassiana (Bs1112) was identified as the teleomorph of Beauveria bassiana using morphology and rDNA internal transcribed spacer sequences. In vitro solid substrate fermentation parameters were evaluated; a rice substrate with 0.01% K+ achieved the maximum spore yield. High-performance liquid chromatography analysis of the bioactive components of C. bassiana spore powder (BC-CBSP), which was extracted using alcohol, showed that concentrations of adenosine were higher than concentrations of inosine, uridine, and cordycepin. The pharmacological impact of BC-CBSP was elucidated in a live animal model, Caenorhabditis elegans, at a range of concentrations from 0.145 to 14.5 mg/mL. The reproductive rate, frequency of head swinging and body bending (indicative of C. elegans locomotion), and lifespan of C. elegans were determined and compared with untreated populations. At the lowest concentration of BC-CBSP, reproduction was slowed but the number of eggs was not reduced. However, at both of the higher concentrations (1.45 and 14.5 mg/mL), BC-CBSP reduced the total number of eggs produced during the 8-day observation period. Furthermore, 0.145 mg/mL of BC-CBSP significantly increased the frequency of head swinging/body bending and the lifespan of C. elegans. This is the first report on the pharmacological effects of C. bassiana spores. Results suggest that low concentrations of BC-CBSP have no negative effects on the reproduction rate of C. elegans but could enhance nervous system activity and extend their lifespan.

The Protective Effect of Adenosine-Preconditioning on Paraquat-Induced Damage in Caenorhabditis elegans.

Adenosine plays an important role in the physiological and pathological conditions of the body by combining different types of adenosine receptors widely distributed in various tissues in the body. In present study, an acute model for paraquat-poisoning in Caenorhabditis elegans was established for quantitative assessment via a time-dose-mortality (TDM) modeling technique with various paraquat doses over 8 hours. Adenosine was first used to precondition at high, medium, and low concentrations and the survival rate of C. elegans was recorded to evaluate adenosine antistress protection against paraquat damage. The results revealed that the TDM model was good for the quantitative assessment of paraquat-poisoning on C. elegans based on the Hosmer-Lemeshow test for homogeneity of modeling (P = .38). The survival rates of adenosine-preconditioned C. elegans have a dose-dependent association with adenosine concentration. At 3000 µM (high concentration) and 300 µM (medium concentration), adenosine-preconditioned C. elegans still had survival rates of 5.38% ± 1.68% and 5.0% ± 1.19% in the subsequent 8 hours observation period. On the contrary, the survival rates of those receiving 30 µM (low concentration) and the 0 µM (unpreconditioned treatment) were zero. To conclude, adenosine preconditioning had protective effects on C. elegans intoxicated with paraquat by decreasing its mortality rate.

Development of a novel isolation unit for entomophthoralean fungi.

Entomophthoralean fungi are important natural enemies of pests and highly co-evolved with their hosts. However, successful isolation of entomophthoralean fungi can be difficult due to their fastidious culture requirements; this is an important obstacle to research on Entomophthorales. In this study, we designed an isolation unit and evaluated it against the conventional 'descending conidia' isolation method. There was no difference in contamination rate between the methods (78% and 76% clean isolations) despite the isolation unit not requiring laminar-flow facilities. Furthermore, more conidia were collected in the new isolation unit than using a standard method. The isolation unit is efficient, convenient and is operational in the field.

Identification of three internal feeders from Korla fragrant pears by quantitative real-time polymerase chain reaction.

Cydia pomonella, Euzophera pyriella Yang, and Grapholitha molesta are destructive pest species of Korla fragrant pears in Xinjiang. They are also quarantine pests of concern in a number of countries. Identification of these small pest larvae by morphological characters is difficult, and misidentifications will influence appropriate quarantine decisions. Here, a 520 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was first amplified and sequenced from each species, and a diagnostic region was observed. Subsequently, the species-specific primer and probe sets of quantitative real-time PCR (qPCR) were designed, which amplified a 114-116 bp fragment of COI genes. This method was validated by amplification DNA extracted from single, multiple, and mixed pest samples. Results indicated that this method allows rapid discrimination and reliable identification of larvae, pupae, and adult specimens of all three species, which could help the international export trading of Korla fragrant pears and related products.

Use of electrical penetration graphs (EPG) and quantitative PCR to evaluate the relationship between feeding behaviour and Pandora neoaphidis infection levels in green peach aphid, Myzus persicae.

A real-time qPCR method was developed, validated, and used to quantity the fungal pathogen, P. neoaphidis, within aphids at different times during infection; colonization rate fitted the Gompertz model well (R2 = 0.9356). Feeding behaviour of P. neoaphidis-infected and uninfected M. persicae were investigated, for the first time, using DC-electrical penetration graphs (DC-EPG) that characterized the waveforms made during different aphid stylet probing periods corresponding to epidermis penetration, salivation and ingestion. In the 6 h following the 12-h incubation period (to achieve infection), there were significant differences in the number of events of Np (non-probing) and C (stylet pathway) between infected and uninfected aphids. However, the difference between total duration of Np and C were not significantly different between infected and uninfected aphids. There were no significant differences in the number of events or total duration of E1 (phloem salivation) or E2 (phloem ingestion) between infected and uninfected aphids. There were significant differences in mean number of events and total duration of the pd waveform (intracellular punctures) in infected and uninfected aphids. In the 16 h prior to death, the same differences in behaviour were observed but they were even more obvious. Furthermore, the total duration time of E2 was significantly greater in uninfected aphids than infected aphids, a change that had not been observed in the first 6 h observation period. In conclusion, qPCR quantification demonstrated 'molecular' colonization levels throughout infection, and EPG data analysis during the two periods (during early infection and then during late infection just prior to death) demonstrated the actual physical effects of fungal infection on feeding behaviour of M. persicae; this has the potential to decrease the aphid's capacity of transmission and dispersal. These studies increase our understanding of the interaction between P. neoaphidis and its host aphid.

Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis.

The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host-pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

[Experimental analysis and countermeasures for EDTA-dependent Pseudothrombocytopenia].

The purpose of this study was to investigate the incidence of clinically common EDTA-dependent pseudo-thrombocytopenia (EDTA-PTCP) and methols for treating this diseese. A total of 1326 cases of thrombocytopenia found at blood routine examination were amalyzed anong 71 535 patients hospitalized in our hospital from January 2010 to May 2013, and 87 cases of PTCP caused EDTA-K anticoagulant were analyzed again by using sodium citrate auticoagulant, at the same time the platelet formation distribution was observed by microscopy of smear with Wright-Giemsa staining. The results showed that the platelet count detected by using EDTA-K anticoagulant in 87 cases was (56 ± 27)×10(9)/L, while the platelet count detected by using sodium citrate was (185 ± 39)×10(9)/L (t = 1.83,P < 0.01). The pseudo-thrombocytopenia incidence cansed by EDTA-K was 0.12%, it was 6.56% for the total number of thrombocytopenia. It is concluded that the incidence of PTCP cansed by EDTA-K is 0.12%, the PTCP is easily misdiagnosed. Therefore, the specimens of platelet count <100 10(9)/L should be tested again. When the platelet aggregation is found, the specimens should be examined again by using sodium citrate in order to avoid misdiagnosis.

Effect of FTY720 on some physiological indexes of Non-Obese Diabetic (NOD) Mice.

The studies were performed to investigate the physiological characteristics of non-obese diabetic (NOD) mice treated with FTY720. At the age of 12 weeks, each mouse was fed with FTY720 or physiological saline once a day for 10 weeks running, and their blood glucose, weight, anti-GAD antibody and organ indexes were determined. No mouse in group FTY720 (NOD mice treated with FTY720) showed diabetic symptoms. The average content of serum anti-GAD antibody in group FTY720 decreased 48.75% (P < 0.01). It was concluded that the spleen, kidney and liver of NOD mice treated with FTY720 shriveled significantly in the progression of diabetes (P < 0.01 or P < 0.05). The body weight of group FTY720 mice was slightly lower than that of the model control (MC) group and these two groups both had less body weight than the normal control (NC) group (P < 0.01). The result of tests of anti-GAD antibody suggested that FTY720 treatment could suppress the anti-GAD response.

[Physiological and biochemical bases of xero-tolerance of fungal biocontrol agents and their application].

Hyphomycetes fungal biocontrol agents usually infect host insects by conidial germination and penetration into insect integument under high humidity condition which is generally the restrictive factor for the application of mycoinsecticides in insect control in the field. It is ideal to produce more xero-tolerant inocula (e.g. conidia) of the agents, although the current formulation technology has helped to some degree to reduce the dependence of fungal formulations on the high humidity. This paper reviewed the advance in basic and applied studies on the physiological and biochemical bases of fungal xero-tolerance and its water activity regulation in the past two decades. Possible approaches to make fungal formulations more xero-tolerant were discussed, with an emphasis on the increased accumulation of biologically compatible solutes such as low-molecular polyols and trehalose to the inocula produced for formulation. More efforts are necessitated to understand the mechanisms involved in molecular biology and in regulating the accumulation of the compatible solutes, and to explore for new technology to enhance the xero-tolerance of the fungal agents through biochemical regulation in mass production.