Selection and evaluation of RT-qPCR reference genes for expression analysis in the tiny egg parasitoid wasp, Trichogramma dendrolimi matsumura (Hymenoptera: Trichogrammatidae)

Huo Liang-xiao,Bai Xue-ping,Che Wu-nan,Ning Su-fang,Lv Lin,Zhang Lisheng,Zhou Jin-cheng,Dong Hui 한국응용곤충학회 2022 Journal of Asia-Pacific Entomology Vol.25 No.2

The egg parasitoid, Trichogramma spp., is an important biological control agent used against a broad range of Lepidopteran pests in agriculture and forestry. The biology of Trichogramma has been studied in details. Further studies should focus on the molecular mechanisms of Trichogramma by qualifying the expression of related genes It is critical to select appropriate reference genes for normalizing RT-qPCR results and establishing a robust method for quantifying target gene expression. This study aimed to identify and validate appropriate reference genes for use in RT-qPCR analysis of Trichogramma dendrolimi. Ten candidate housekeeping genes, namely betaactin (ACTIN), forkhead box O (FOXO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock pro tein 90 (HSP90), ribosomal protein L10a (RPL10a), L18 (RPL18), L28 (RPL28), S13 (RPS13), S15 (RPS15), and superoxide dismutase (SOD), were tested for their suitability as reference genes for developmental stage (3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th day after parasitization), tissue (head, thorax, and abdomen of adults), sex of adults (male and female), and temperature (17℃, 25℃, and 32℃). According to the GeNorm analysis, a robust analysis should involve using an appropriate combination of reference genes, namely, at least three genes for different development stages, two genes for different tissues, two genes for different sex, and two genes for different temperatures, respectively. According to the RelFinder method by the integrated results of GeNorm, NormFinder, BestKeeper, and the ΔCt method, we identified the developmental stage-specific reference genes SOD, GAPDH, and ACTIN; tissue-specific reference genes RPL18 and RPS15; sex-specific reference genes RPL18 and SOD; and temperature-specific reference genes RPL18 and RPL10a. This study provides a standardized procedure for the quantification of gene expression in T. dendrolimi and will be helpful for future biological control programs using Trichogramma wasps.

Radiosensitivity Enhancement by Arsenic Trioxide in Conjunction with Hyperthermia in the EC-1 Esophageal Carcinoma Cell Line

Cui, Yan-Hui,Liang, Hai-Jun,Zhang, Qing-Qin,Li, Si-Qing,Li, Xiao-Rui,Huo, Xiao-Qing,Yang, Qing-Hui,Li, Wei-Wei,Gu, Jian-Fa,Hua, Qin-Liang,Lu, Ping,Miao, Zhan-Hui Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.4

Objective: To explore the effect on radiosensitivity of arsenic trioxide ($As_20_3$) in conjunction with hyperthermia on the esophageal carcinoma EC-1 cell line. Method: Inhibition of EC-1 cell proliferation at different concentrations of $As_20_3$ was assessed using the methyl thiazolyl blue colorimetric method (MTT method), with calculation of $IC_{50}$ value and choice of 20% of the $IC_{50}$ as the experimental drug concentration. Blank control, $As_20_3$, hyperthermia, radiotherapy group, $As_20_3$ + hyperthermia, $As_20_3$ + radiotherapy, hyperthermia + radiotherapy and $As_20_3$ + hyperthermia + radiotherapy groups were established, and the cell survival fraction (SF) was calculated from flat panel colony forming analysis, and fitted by the 'multitarget click mathematical model'. Flow cytometry (FCM) was used to detect changes in cell apoptosis and the cell cycle. Results: $As_20_3$ exerted inhibitory effects on proliferation of esophageal carcinoma EC-1 cells, with an $IC_{50}$ of 18.7 ${\mu}mol/L$. After joint therapy of $As_20_3$ + hyperthermia + radiotherapy, the results of FCM showed that cells could be arrested in the $G_2$/M phase, and as the ratio of cells in $G_0/G_1$ and S phases decreased, cell death became more pronounced. Conclusion: $As_20_3$ and hyperthermia exert radiosensitivity effects on esophageal carcinoma EC-1 cells, with synergy in combination. Mechanistically, $As_20_3$ and hyperthermia mainly influence the cell cycle distribution of EC-1 esophageal carcinoma cells, decreasing the repair of sublethal damage and inducing apoptosis, thereby enhancing the killing effects of radioactive rays.

One simple, rapid and economical method for ploidy detection of Trichogramma dendrolimi Matsumura (Hymenoptera Trichogrammatidae)

Liu Quan-quan,Zhang Chen,Zhou Jin-cheng,Dong Qian-jin,Huo Liang-xiao,Dong Hui 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.2

Ploidy diversity provides valuable scientific information, thus making the detection technique of ploidy important. However, traditional methods of cytological observation and flow cytometry are either laborious or expensive. We here report a simple and rapid, effective and economical quantitative PCR (qPCR) approach to determine the ploidy of a parasitoid species Trichogramma dendrolimi Matsumura, an economically important biocontrol agent. We applied a mitochondrial gene cytochrome oxidase (COI) and a nuclear gene forkhead to evaluate the mitochondrial number per nuclear copy in a thelytokous Wolbachia-infected strain of T. dendrolimi and its bisexual uninfected counterparts. The 2 −ΔCq values calculated from C q values which resulted from qPCR experiments were significantly larger in haploid males than that in diploid females. Haploid males possessed about 2.69 times mitochondrial number per nuclear copy as diploid females. Not a single significant difference was found between diploid females from thelytokous and bisexual strains. Based on the differences in relative mitochondrial content, we were allowed to distinguish between haploid males and diploid females. Moreover, the number of mitochondria significantly decreased with higher ploidy level but was not affected by Wolbachiainfection. Our study supplied an available tool to investigate the ploidy diversity in sex determination of T. dendrolimi and thelytokous manipulation of Wolbachia, which is the crucial step to further study their underlying mechanisms. This will in turn contribute to the biocontrol efficiency by enhancing the female production and hence the parasitism rate.

Increased Serotonin Signaling Contributes to the Warburg Effect in Pancreatic Tumor Cells Under Metabolic Stress and Promotes Growth of Pancreatic Tumors in Mice

Jiang, Shu-Heng,Li, Jun,Dong, Fang-Yuan,Yang, Jian-Yu,Liu, De-Jun,Yang, Xiao-Mei,Wang, Ya-Hui,Yang, Min-Wei,Fu, Xue-Liang,Zhang, Xiao-Xin,Li, Qing,Pang, Xiu-Feng,Huo, Yan-Miao,Li, Jiao,Zhang, Jun-Feng Elsevier 2017 Gastroenterology Vol.153 No.1

<P><B>Background & Aims</B></P> <P>Desmoplasia and poor vascularity cause severe metabolic stress in pancreatic ductal adenocarcinomas (PDACs). Serotonin (5-HT) is a neuromodulator with neurotransmitter and neuroendocrine functions that contributes to tumorigenesis. We investigated the role of 5-HT signaling in the growth of pancreatic tumors.</P> <P><B>Methods</B></P> <P>We measured the levels of proteins that regulate 5-HT synthesis, packaging, and degradation in pancreata from Kras<SUP>G12D/+</SUP>/Trp53<SUP>R172H/+</SUP>/Pdx1-Cre (KPC) mice, which develop pancreatic tumors, as well as in PDAC cell lines and a tissue microarray containing 81 human PDAC samples. We also analyzed expression levels of proteins involved in 5-HT synthesis and degradation by immunohistochemical analysis of a tissue microarray containing 311 PDAC specimens, and associated expression levels with patient survival times. 5-HT level in 14 matched PDAC tumor and non-tumor tissues were analyzed by ELISA. PDAC cell lines were incubated with 5-HT and cell survival and apoptosis were measured. We analyzed expression of the 5-HT receptor HTR2B in PDAC cells and effects of receptor agonists and antagonists, as well as HTR2B knockdown with small hairpin RNAs. We determined the effects of 5-HT stimulation on gene expression profiles of BxPC-3 cells. Regulation of glycolysis by 5-HT signaling via HTR2B was assessed by immunofluorescence and immunoprecipitation analyses, as well as by determination of the extracellular acid ratio, glucose consumption, and lactate production. Primary PDACs, with or without exposure to SB204741 (a selective antagonist of HTR2B), were grown as xenograft tumors in mice, and SB204741 was administered to tumor-bearing KPC mice; tumor growth and metabolism were measured by imaging analyses.</P> <P><B>Results</B></P> <P>In immunohistochemical analysis of a tissue microarray of PDAC specimens, increased levels of TPH1 and decreased level of MAOA, which regulate 5-HT synthesis and degradation, correlated with stage and size of PDACs and shorter patient survival time. We found levels of 5-HT to be increased in human PDAC tissues compared with non-tumor pancreatic tissues, and PDAC cell lines compared with non-transformed pancreatic cells. Incubation of PDAC cell lines with 5-HT increased proliferation and prevented apoptosis. Agonists of HTR2B, but not other 5-HT receptors, promoted proliferation and prevented apoptosis of PDAC cells. Knockdown of HTR2B in PDAC cells, or incubation of cells with HTR2B inhibitors, reduced their growth as xenograft tumors in mice. We observed a correlation between 5-HT and glycolytic flux in PDAC cells; levels of metabolic enzymes involved in glycolysis, the phosphate pentose pathway, and hexosamine biosynthesis pathway increased significantly in PDAC cells following 5-HT stimulation. 5-HT stimulation led to formation of the HTR2B–LYN–p85 complex, which increased PI3K–Akt–mTOR signaling and the Warburg effect by increasing protein levels of MYC and HIF1A. Administration of SB204741 to KPC mice slowed growth and metabolism of established pancreatic tumors and prolonged survival of the mice.</P> <P><B>Conclusions</B></P> <P>Human PDACs have increased levels of 5-HT, and PDAC cells increase expression of its receptor, HTR2B. These increases allow for tumor glycolysis under metabolic stress and promote growth of pancreatic tumors and PDAC xenograft tumors in mice.</P>