Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus / 中国热带医学

@#Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

COVID-19 surveillance via wastewater:Introduction of wastewater-based epidemiology / 西安交通大学学报(医学版)

Since the outbreak of the COVID-19 global pandemic in 2019, monitoring COVID-19 infection status and trend through wastewater, known as wastewater-based epidemiology (WBE), has been widely used in many countries and regions. WBE consists of five steps:wastewater sample collection, viral concentration, viral nucleic acid extraction, quantification of virus using quantitative RT-PCR, and dissemination of the wastewater surveillance results. This method could be used for early warning of COVID-19 outbreak in a population, monitoring COVID-19 distributions and epidemic trend, prediction of COVID-19 prevalence rate, understanding of temporal trend of SARS-COV-2 variants, and simultaneous surveillance of multiple pathogens. WBE and clinical surveillance can be used concurrently and the former is a good complement to the latter.

Efeito da irrigação ultrassônica e da medicação intracanal com hidróxido de cálcio na quantidade e no metabolismo de bactérias que persistiram após o preparo dos canais radiculares de dentes com periodontite apical / Effect of ultrasonic irrigation and calcium hydroxide intracanal medication on the levels and metabolism of bacteria that persisted after root canal preparation of teeth with apical periodontitis

Estudos moleculares ressaltam as limitações do protocolo endodôntico tradicional em eliminar bactérias dos canais radiculares. Apesar do preparo químico-cirúrgico (PQC) promover uma drástica redução bacteriana, muitos canais continuam infectados após essa etapa do tratamento. Dessa forma, estudos apontam para a necessidade de complementação técnica para potencializar a desinfecção dos canais radiculares após o PQC. Assim, o objetivo deste estudo clínico foi avaliar, por métodos moleculares baseados em DNA e RNA, o efeito dos métodos complementares ao preparo na desinfecção dos canais radiculares. Coletas microbiológicas dos canais de 20 dentes unirradiculares com periodontite apical foram feitas em diferentes etapas do tratamento endodôntico: previamente ao preparo (S1); após o PQC realizado com sistema Reciproc associado à irrigação com NaOCl 2,5% (S2); após a irrigação ultrassônica passiva, denominada PUI (S3); e após a medicação intracanal à base de hidróxido de cálcio (S4). As amostras foram submetidas à extração de DNA e RNA. O RNA foi submetido à reação de transcrição reversa (RT-PCR) para confecção da fita dupla de DNA complementar (cDNA). DNA e cDNA foram submetidos a reações de qPCR, com iniciadores universais para a região 16S rRNA do domínio Bacteria. A atividade metabólica das bactérias foi verificada através da relação entre os níveis de rRNA e rDNA determinados pelos ensaios de qPCR. Os dados foram analisados pelo teste de Wilcoxon para amostras pareadas (p < 0,05). As amostras S1 dos 20 casos apresentaram altos níveis de rDNA (mediana: 1,25 x 105, intervalo 1,83 x 104 - 9,2 x 106) e rRNA bacteriano (mediana: 5,47 x 105, intervalo 7,8 x 104 - 5,95 x 107). Dezessete canais (85%) apresentaram reações qPCR positivas para rDNA nas amostras pós-preparo (S2). A redução de rDNA após o preparo foi estatisticamente significativa (p = 0,0003), com mediana de 2,5 x 104 (intervalo 2,26 x 103 - 9,52 x 104) cópias de rDNA em S2. Por sua vez, os níveis de rRNA (mediana: 7,84 x 104, intervalo 2,91 x 103 - 1,09 x 106) foram maiores que os níveis de rDNA (p = 0,01), sugerindo que essas bactérias estavam metabolicamente ativas em S2. Após a PUI, o número de amostras S3 com resultados positivos para rDNA caiu para 12, representando uma redução significativa em relação às amostras S2 (p = 0,008). Além disso, a PUI promoveu uma redução significativa dos níveis de rDNA (mediana 2,94 x 103, intervalo 2,70 x 103 - 1,09 x 105) em relação à amostras S2 (p = 0,01). Na análise baseada em rRNA, os níveis em S3 (mediana: 03 x 104, intervalo 1,82 x 103 - 1,39 x 105) não apresentaram diferença significativa em comparação aos níveis de rDNA (p = 0,07), sugerindo que houve uma redução do metabolismo bacteriano após a PUI. Em S4, o número de casos positivos para rDNA bacteriano (n = 13) e os níveis de rDNA (mediana: 3,73 x 104, intervalo 1,98 x 103 - 3,21 x 105) foram ligeiramente maiores quando comparados aos valores das amostras S3, porém sem diferenças significativas. Entretanto, os níveis de rRNA (mediana: 1,08 x 105, intervalo 3,41 x 103 - 1,60 x 106) foram maiores que os de rDNA (p = 0,02) nas amostras S4, sugerindo que as bactérias retomaram sua atividade metabólica apesar do uso da medicação intracanal. Portanto, foi possível concluir que a irrigação ultrassônica passiva contribuiu para a desinfecção dos canais radiculares, promovendo uma redução do número e do metabolismo de bactérias. Por outro lado, as bactérias persistiram ativas nos canais radiculares após o uso do hidróxido de cálcio como medicação intracanal em dentes com periodontite apical.

Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli

Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.

Over-expression of dehydroascorbate reductase enhances oxidative stress tolerance in tobacco

Background: Ascorbic acid (Asc) is one of the most abundant antioxidants and it serves as a major contributor to protect plants against oxidative damage. Plants use two enzymes that participate in the metabolic recycling of Asc. One of these two enzymes is dehydroascorbate reductase (DHAR). It directly regenerates Asc from its oxidized state and thus prevents Asc from being irreversibly hydrolyzed to 2, 3-diketogulonic acid. This study aimed to examine whether over-expression of DHAR leads to an enhanced oxidative stress tolerance in tobacco plants. Results: In this study, we functionally characterized a novel JcDHAR gene from Jatropha curcas and found via quantitative RT-PCR analysis that JcDHAR can be induced with H2O2, salt and PEG stresses. The DHAR activities of transgenic tobacco plants increased from 2.0 to 5.3 fold compared to wild-type plants. As a result, the transgenic plants displayed enhanced tolerance to oxidative stress. Conclusions: Our results indicate that JcDHAR expression can effectively enhance the tolerance to oxidative stress in plants.

Synthesized flavanoid-derived ligand reduced dengue virus type-2 replication in vitro

Objective To investigate the antiviral property of a lead ligand, YK51 that was synthesized based on the flavanoid of a natural product toward dengue virus type-2 (DENV2) replication. Methods cRNA was isolated from HepG2 cells inoculated with 1 000 median tissue culture infective dose of DENV2 and treated with different doses of the ligand followed by RT-PCR to quantify the virus gene copies. Confocal microscopy of actin and tubulin redistribution was also performed. Results The quantitative RT-PCR result showed reduction of the DENV2 gene copies as the ligand concentration was increased. The confocal microscopy result showed increase in the tubulin intensity (79.6%) of infected BHK21 cells treated with the ligand, compared with the non-treated cells (54.8%). The 1.5-fold increase in the intensity of tubulin suggested that the ligand inhibitory effect stabilized the cellular microtubule structure. Conclusions The synthesized ligand YK51 reduced DENV2 viral load by inhibiting virus replication thus is highly potential to be developed as antiviral agent.

Cloning and expression of geranyl pyrophosphate synthase gene in Swertia mussotii / 中草药

Objective: To clone the geranyl pyrophosphate synthase gene from Swertia mussotii (SmGPPS), analyze the bioinformation of SmGPPS, and perform the gene expression. Methods: According to the SmGPPS gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector pET-28a-SmGPPS was constructed and transformed into Escherichia coli BL-21 (DE3) for expression under 37℃ and induced by 1 mmol/L IPTG. The relative expression of gene SmGPPS in the leaf, stem, and flower of S. mussotii was also studied. Results: The results showed that SmGPPS cDNA complete sequences had a length of 1 119 bp encoding 372 amino acid residues. And the protein secondary and tertiary structures were analyzed and forecasted. The SmGPPS protein shared high identity with other GPPS proteins of plants. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Relative RT-PCR analysis indicated that SmGPPS showed the highest transcript abundance in the leaf. Conclusion: This work will provide a foundation for further functional research of SmGPPS protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.

Synthesized flavanoid-derived ligand reduced dengue virus type-2 replication in vitro

Objective: To investigate the antiviral property of a lead ligand, YK51 that was syn-thesized based on the flavanoid of a natural product toward dengue virus type-2 (DENV2) replication. Methods: cRNA was isolated from HepG2 cells inoculated with 1000 median tissue culture infective dose of DENV2 and treated with different doses of the ligand followed by RT-PCR to quantify the virus gene copies. Confocal microscopy of actin and tubulin redistribution was also performed. Results: The quantitative RT-PCR result showed reduction of the DENV2 gene copies as the ligand concentration was increased. The confocal microscopy result showed increase in the tubulin intensity (79.6%) of infected BHK21 cells treated with the ligand, compared with the non-treated cells (54.8%). The 1.5-fold increase in the intensity of tubulin suggested that the ligand inhibitory effect stabilized the cellular microtubule structure. Conclusions: The synthesized ligand YK51 reduced DENV2 viral load by inhibiting virus replication thus is highly potential to be developed as antiviral agent.

Comparison of hPepT1 transfected MDCK cells to hPepT1 transfected HeLa cells / 中国药理学通报

Aim To screen a more suitable transfection recep-tor,and improve the efficiency of constructing cell lines highly expressing human peptide transporters 1 (hPepT1 ).Methods The recombinant plasmid pcDNA3.1 (+)-hPepT1 was transfect-ed into MDCK cells and HeLa cells by LipofectamineTM 2000 transfection reagent,respectively.The monoclonal cells were se-lected and cultured.Expression of hPepT1 mRNA and protein were determined by qRT-PCR and Western blot,respectively. The uptake capacity of Glysar in transfected cells was examined. Results Compared with wild type cells,the expression of hPepT1 and the uptake of Glysar in transfected MDCK cells and HeLa cells significantly increased (P <0.05).Although the up-take of Glysar in HeLa cells was higher than that of MDCK cells,on the contrary,the expression of hPepT1 and the uptake of Glysar in MDCK-hPepT1 cells was higher than that of HeLa-hPepT1 cells.Conclusion MDCK cells may serve as a more suitable transfected receptor for the construction of a cellular model with high expression of hPepT1 ,which would make the construction of a cell model highly expressing hPepT1 more effi-cient.

NaCl stress impact on the key enzymes in glycolysis from Lactobacillus bulgaricus during freeze-drying

Abstract The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level.

Swim training and the genetic expression of adipokines in monosodium glutamate-treated obese rats

Objective The aim of this study was to evaluate the genetic expression of adipokines in the adipocytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity.Materials and methods Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adipokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed.Conclusions In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.

Comparison between the quantitative and semi-quantitative fluorescence RT-PCR assays for the detection of XDH/XO mRNA expression in different organ tissues of rhesus monkey / 中国比较医学杂志

Objective To analyze the differences between the semi-quantitative RT-PCR and real time quantitative fluorescence RT-PCR assays for detecting XDH/XO mRNA expression in various organ tissues of rhesus monkey, and provide useful reference in methodology of experimental studies.Total RNA was extracted from the myocardium, kidney, testis, skin, and liver tissues, respectively, for detecting XDH/XO mRNA expression in rhesus monkey by semi-quantitative RT-PCR and real time quantitative fluorescence RT-PCR assays.The sensitivity and specificity of the two assays were compared with each other using the same primer sequences and reference genes.Results The expression of XDH/XO mRNA in different organ tissues were detected by both the two PCR assays.The sensitivity of quantitative fluorescence real-time RT-PCR for the XDH/XO mRNA expression in the liver tissue was 39 times higher than that by semi-quantitative RT-PCR.Conclusions Both the quantitative and semi-quantitative fluorescence RT-PCR assays can be used to detect the expression of XDH/XO mRNA in different organ tissues of rhesus monkey.The sensitivity of quantitative fluorescence real-time PCR assay is more sensitive than that of the semi-quantitative RT-PCR assay.

Molecular characteristics, recombinant expression and activity detection of OsGSTL1 from rice

The mRNA of OsGSTL1 was detected in the roots and leaves of rice plants at seedling and tillering stages, and their roots, leaves and panicles at the heading stage. The full-length open reading frame of OsGSTL1 cDNA was 732 bp and encoded a putative polypeptide of 243 amino acids with a calculated molecular mass of 27.30 kDa and a theoretical pI of 5.50. The protein sequences of OsGSTL1 exhibited typical feature of the lambda class GST, which contained the conserved domain "GST_C_Lambda" in C-terminal alpha helical domain and a highly conserved Cys42 in active center. In silico predictions showed that the OsGSTL1 protein was strongly hydrophilic. The phylogenetic analysis revealed OsGSTL1 belonged to monocots subgroup and was closer to IN2-1 of Z. may. The OsGSTL1 gene was cloned into pYTV vector and was introduced into yeast strain PEP4. Western blot analysis showed that the exogenous OsGSTL1 was expressed in the transformed yeast. The GST activity of the crude extracts of yeast showed that the OsGSTL1 transgenic yeast had higher levels of GST activities than the control yeasts. These findings suggested that the OsGSTL1 was a glutathione S-transferase and could play an important role during the growth and development processes in rice.

Transcriptional profiling of genes involved in n-hexadecane compounds assimilation in the hydrocarbon degrading Dietzia cinnamea P4 strain

The petroleum-derived degrading Dietzia cinnamea strain P4 recently had its genome sequenced and annotated. This allowed employing the data on genes that are involved in the degradation of n-alkanes. To examine the physiological behavior of strain P4 in the presence of n-alkanes, the strain was grown under varying conditions of pH and temperature. D. cinnamea P4 was able to grow at pH 7.0-9.0 and at temperatures ranging from 35 ºC to 45 ºC. Experiments of gene expression by real-time quantitative RT-PCR throughout the complete growth cycle clearly indicated the induction of the regulatory gene alkU (TetR family) during early growth. During the logarithmic phase, a large increase in transcriptional levels of a lipid transporter gene was noted. Also, the expression of a gene that encodes the protein fused rubredoxin-alkane monooxygenase was enhanced. Both genes are probably under the influence of the AlkU regulator.

Real-time fluorescent quantitative reverse transcription polymerαse chain reaction in detecting expression of ERα, PR and Her2 mRNA in breast cancer tissues / 第二军医大学学报

Objective To evaluate the clinical value of real-time fluorescent quantitative reverse transcription polym ERαse chain reaction (qRT-PCR) in detecting expression of estrogen receptor alpha (.ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HerZ) genes in breast cancer tissues. Methods Totally 48 breast cancer tissues and 28 benign breast tumor tissues (control) were obtained from patients undergoing surgery in our hospital during Mar. 2010 and Oct. 2010. The expression of ERα, PR and Her2 protein was examined by immunohistochemistry (IHC) in breast cancer tissues and the expression levels of ERα, PR and HerZ mRNA were detected by real-time qRT-PCR in breast cancer tissues and benign breast tumor tissues. The values of these two methods in diagnosis of breast cancer were evaluated. Results The expressions of ERα and HerZ mRNA were significantly higher in the breast cancer tissues than in the controls (P0. 05). Real-time qRT-PCR in detecting ERα, PR and Her2 mRNA expression had similar capability with IHC method in evaluating the sensitivity, specificity of endocrine thERαpy; moreover, the two methods also had a consistent pathological diagnosis rates (P>0. 05). Conclusion ERα, PR and Her2 genes are important predictive markers for endocrine thERαpy or targeted thERαpy of breast cancer. Real-time qRT-PCR method can be used for clinical detection and research of ERα, PR and Her2 mRNA.

Expression of IMD, CRLR, RAMP1, RAMP2 and RAMP3 in cancer tissues of patients with non-small cell lung cancer and their clinical significance / 肿瘤研究与临床

Objective To investigate the expression and significance of intermedin (IMD) and its.receptors CRLR,RAMP1,RAMP2 and RAMP3 in cancer tissues of patients with non-small cell lung cancer.Methods The mRNA gene expressions of IMD,CRLR,RAMP1,RAMP2 and RAMP3 were detected by realtime quantitative RT-PCR in cancerous and para-cancerous tissues from 27 patients with lung cancer.Results Real-time quantitative PCR detection results showed that the expression of IMD,CRLR,RAMP1,RAMP2 and RAMP3 in cancer tissues were [(59±7.9)×10-8,(96±2.7)×10-6,(29±3.9)×10-9,(14±2.6)×10-6,(65±1.1)×10-6]which were higher than those in adjacent tissues[(40±4.7)×10-10,(21 ±3.9)×10-6,(53±7.8)×10-10,(64±1.9)×10-8,(36±1.3)×10-9] to some extent (all P ＜ 0.05); the higher expression of RAMP3 was found is higher expressions than RAMP1 and RAMP2 in cancer tissues (all P ＜ 0.05).Conclusion The expressions of IMD and its receptors in cancer tissues are higher than those in paracancerous tissues.IMD may play an important role in the development of cancer by activate RAMP3 which is the most high expressed receptor in cancer tissues.Therefore,it might be helpful for the investigation of new gene thereapy in non-small cell lung cancer.

Quantitative Evaluation of Viability- and Apoptosis-Related Genes in Ascaris suum Eggs under Different Culture-Temperature Conditions

Ascaris suum eggs are inactivated by composting conditions; however, it is difficult to find functional changes in heat-treated A. suum eggs. Here, unembryonated A. suum eggs were incubated at 20degrees C, 50degrees C, and 70degrees C in vitro, and the gene expression levels related to viability, such as eukaryotic translation initiation factor 4E (IF4E), phosphofructokinase 1 (PFK1), and thioredoxin 1 (TRX1), and to apoptosis, such as apoptosis-inducing factor 1 (AIF1) and cell death protein 6 (CDP6), were evaluated by real-time quantitative RT-PCR. No prominent morphological alterations were noted in the eggs at 20degrees C until day 10. In contrast, the eggs developed rapidly, and embryonated eggs and hatched larvae began to die, starting on day 2 at 50degrees C and day 1 at 70degrees C. At 20degrees C, IF4E, PFK1, and TRX1 mRNA expression was significantly increased from days 2-4; however, AIF1 and CDP6 mRNA expression was not changed significantly. IF4E, PFK1, and TRX1 mRNA expression was markedly decreased from day 2 at 50degrees C and 70degrees C, whereas AIF1 and CDP6 mRNA expression was significantly increased. The expressions of HSP70 and HSP90 were detected for 9-10 days at 20degrees C, for 3-5 days at 50degrees C, and for 2 days at 70degrees C. Taken together, incremental heat increases were associated with the rapid development of A. suum eggs, decreased expression of genes related to viability, and earlier expression of apoptosis-related genes, and finally these changes of viability- and apoptosis-related genes of A. suum eggs were associated with survival of the eggs under temperature stress.

Expression of the survivin-2B splice variant related to the progression of colorectal carcinoma / 대한외과학회지

PURPOSE: Recently, two alternatively spliced survivin variants, survivin-DeltaEx3 and survivin-2B, were identified in a single copy of the survivin gene. It has been reported that the expressions of survivin splice variants significantly correlates with the clinical results in many types of human carcinoma. We investigated the transcription levels of survivin and its splice variants in human colorectal carcinomas, and analyzed correlations between survivin expression levels and clinicopathologic features. METHODS: We used Western blot and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) to analyze the protein and mRNA expression levels of survivin variants in 51 colorectal carcinomas. The quantitative RT-PCR was performed using primer pairs specific for survivin and each of its splice variants, then normalized for the gene that encodes glyceraldehydes-3-phosphate dehydrogenase. RESULTS: In Western blotting, the protein levels of survivin were higher in the tumor tissue than in normal tissue. The expression of survivin, survivin-2B and survivin-DeltaEx3 mRNA was present in 96%, 64.7%, and 82.4% of the samples, respectively. When the pathologic parameters were compared, colorectal cancers of advanced pT stages showed significant decrease in survivin-2B mRNA expression by the quantitative RT-PCR (P < 0.001). CONCLUSION: The decreased expression of survivin-2B might be related to tumor progression in colorectal cancers. This finding indicates that alternatively spliced variants of survivin may be involved in refining the functions of survivin during tumor progression.

Classification of Biological Effect of 1,763 MHz Radiofrequency Radiation Based on Gene Expression Profiles

Radiofrequency (RF) radiation might induce the transcription of a certain set of genes as other physical stresses like ionizing radiation and UV. To observe transcriptional changes upon RF radiation, we exposed WI-38, human lung fibroblast cell to 1763 MHz of mobile phone RF radiation at 60 W/kg of specific absorption rate (SAR) for 24h with or without heat control. There were no significant changes in cell numbers and morphology after exposure to RF radiation. Using quantitative RT-PCR, we checked the expression of three heat shock protein (HSP) (HSPA1A, HSPA6 and HSP105) and seven stress-related genes (TNFRSF11B, FGF2, TGFB2, ITGA2, BRIP1, EXO1, and MCM10) in RF only and RF/HS groups of RF-exposed cells. The expressions of three heat shock proteins and seven stress-related genes were selectively changed only in RF/HS groups. Based on the expression of ten genes, we could classify thermal and non-thermal effect of RF-exposure, which genes can be used as biomarkers for RF radiation exposure.

Clinical significance of blood micrometastasis in pN0 esophageal squamous cell carcinoma / 肿瘤研究与临床

Objective To explore the clinical significance and the impact on prognosis of blood micrometastasis in the patients with pN0 esophageal squamous cell carcinoma. Methods Total RNA was extracted with TRIzol and mRNA was transcribed reversely into cDNA. RT-PCR was used to detect MMP-7 mRNA and hTERT mRNA in blood. △△Ct sample values were calculated with post-operative follow-up of 3 month, 6 month, 12 month. Results Statistical results suggested that blood micrometastasis was related to differentiation grade and pTNM staging (P=0.000, P=0.000 respectively), but not to age, sex, length of turnout (P0.05). Follow-up results suggested that the degree of invasion and tumor metastasis (recurrence) was no correlation; follow-up to 6 month and 12 month, tumor metastasis (recurrence) was associated with blood micrometastasis, and follow-up to 12 month, compared with the tumor metastasis (recurrence) probability of blood micrometastasis-positive patients and negative patients, the former was as 6.44 times as the latter. (OR=6.440, 95 % CI 1.547-26.822). Conclusion Blood micrometastasis testing is of great significance to early diagnosis and prognosis judgment in pN0 esophageal squamous cell carcinoma patients.