AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl trans...AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.展开更多
Risk of clinically significant prostate adenocarcinoma (CAP) varies worldwide,although there is a uniform prevalence of latent disease. A hormone-responsive tissue,the prostate possesses the metabolizing capacity to...Risk of clinically significant prostate adenocarcinoma (CAP) varies worldwide,although there is a uniform prevalence of latent disease. A hormone-responsive tissue,the prostate possesses the metabolizing capacity to biotransform a variety of environmental procarcinogens or endogenous hormones. Whether such metabolizing capacity or estrogen receptor (ER) status underlies these demographic differences in susceptibility to CaP remains unclear. With appropriate ethical permission,verified-benign tissues were obtained following transurethral resection of the prostate from a high-risk region (n = 12 UK-resident Caucasians) and a typically low-risk region (n = 14 India-resident Asians). Quantitative gene expression analysis was employed for cytochrome P450 (CYP)1B1,N-acetyltransferase (NAT)1,NAT2,catechol-O-methyl transferase ( COMT),sulfotransferase ( SULT) 1A1,ERα,ERβ and aromatase (CYP To quantify the presence or absence of CYP1B1,ERα or ERβ,and to identify ther in situ localization,immunohistochemistry was carried out. The two cohorts had reasonably well-matched serum levels of prostate-specific antigen or hormones. Expression levels for the candidate genes investigated were similar.However,clear differences in protein levels for CYP1B1 and ERβ were noted. Staining for CYP1B1 tended to be nuclear-associated in the basal glandular epithelial cells,and in UK-resident Caucasian tissues was present at a higher (P = 0.006) level compared with that from India-resident Asians. In contrast,a higher level of positive ERβ staining was noted in prostates from India-resident Asians. These study findings point to differences in metabolizing capacity and ER status in benign prostate tissues that might modulate susceptibility to the emergence of clinically significant CaP in demographically distinct populations.展开更多
The sheep genome harbours approximately 20 copies of endogenous beta-retroviruses (enJSRVs), and circumstantial evidence suggests that enJSRVs might play a role in mammalian reproduction, particularly placental morp...The sheep genome harbours approximately 20 copies of endogenous beta-retroviruses (enJSRVs), and circumstantial evidence suggests that enJSRVs might play a role in mammalian reproduction, particularly placental morphogenesis. This study was aimed to assess the expression of mRNAs of an enJSRV and its receptor, HYAL2, in the uterus and conceptuses of Mongolian ewes throughout gestation, using real-time reverse transcription polymerase chain reaction and in situ hybridization analysis. The results showed that enJSRV and HYAL2 mRNAs were found to be expressed throughout gestation in the endometrium, chorion, placenta, and conceptus. The enJSRV mRNA was most abundant in the placenta on day 90 of pregnancy, in the endometrium on day 30 and 50, and in the chorion on day 70 and 110. However, HYAL2 mRNA was most abundant in the endometrium on day 30. These differences were all significantly different from each other (P〈0.01). In situ hybridization showed that enJSRV and HYAL2 mRNAs were specifically expressed in endometrial luminal epithelium and glandular epithelium, trophoblastic giant binucleated cells (BNCs), endometrial caruncles, placental cotyledons, stroma, trophectoderm, as well as multinucleated syncytia of the placenta and blood vessel endothelial cells. Collectively, little is known about the molecular mechanisms by which trophoblastic differentiation and multinucleated syncytia formation are regulated by enJSRVs. However, the temporal and spatial distributions of enJSRV expression in the uterus and conceptus indicate that differentiation of BNCs and the formation of a multinucleated syncytiotrophoblast involve enJSRV and possibly its cellular receptor, HYAL2. Therefore, enJSRV and HYAL2 appear to play important roles in the female reproductive physiology in this breed of sheep.展开更多
BACKGROUND: Interstitial collagenase has been considered as an essential enzyme for collagenolysis in liver fi-brosis, because type Ⅰ and Ⅲ collagens increase predominantly in liver fibrosis. The present study aimed...BACKGROUND: Interstitial collagenase has been considered as an essential enzyme for collagenolysis in liver fi-brosis, because type Ⅰ and Ⅲ collagens increase predominantly in liver fibrosis. The present study aimed to demonstrate the gene expression of matrix metalloproteinases-13 (MMP-13) in the progressive phases of ethanol induced experimental liver fibrosis in rats. METHODS: Thirty-four Sprague-Dawley rats were randomly divided into two groups. The experimental group (24 rats) was given ethanol (44% , 7 g/kg) every day and the control group (10) was given normal saline. Liver samples were harvested from experimental rats at 4, 12 and 24 weeks respectively. The kinetics of MMP-13 mRNA expression was assayed by semi-quantity reverse transcriptase-poly-merase chain reaction (RT-PCR). RESULTS: In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR (0.24±0.41). The gene expression of MMP-13 was increased in the liver of the rats treated with ethanol for 4 weeks (0. 62 ±0. 54), but it was not considered statistically significant (P >0.05). And the livers from 12-week-treated rats showed a marked mRNA expression(1.65 ±0.47, P <0. 01). Once fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39±0.25). CONCLUSION: MMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, and it was induced in a distinct time frame.展开更多
Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential.Structural and functional characterization of drought stress-induced genes has contributed to a bette...Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential.Structural and functional characterization of drought stress-induced genes has contributed to a better understanding of how plants respond and adapt to the drought stress.In the present study,differential display technique was employed to study the gene expression of rice plants at the reproductive stage that were subjected to drought stress by withholding water,Pseudomonas fluorescens strain(Pf1) treated plants subjected for drought stress by withholding water and control(well-watered).Differentially expressed c DNAs of six genes(COX1,PKDP,b ZIP1,AP2-EREBP,Hsp20 and COC1) were identified,cloned and sequenced.Real-time q PCR analysis showed that all the six genes were upregulated in drought-stressed plants treated with Pf1.This revealed that the remarkable influence of Pf1 colonization leads to drought tolerance at the reproductive stage.These results showed that high levels of gene expression in plants lacking adequate water can be remarkably influenced by Pf1 colonization,which might be a key element for induced systemic tolerance by microbes.展开更多
Summary: In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor β (TGF-β), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemic...Summary: In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor β (TGF-β), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical methods were used for detection of TGF-β mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-β immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-β, and LEC could be affected by TGF-β through autocrine action.展开更多
BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adul...BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adults clear virus from primary HBV infection, so the difference of gene expression between ASCs and normal adults determines the differential outcomes. To identify differentially expressed genes in ASCs compared to normal adults, we used suppression subtractive hybridization to compare gene expression. METHODS: cDNA subtracted libraries were constructed by suppression subtracted hybridization from peripheral blood monocytes of ASCs and normal adults, the subtracted clones were prescreened by dot blot hybridization, and the levels of genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: One hundred and two and 96 positive clones were acquired from the A-N and N-A libraries, respectively, and 14 and 11 clones were identified by dot blot hybridization in the A-N and N-A libraries. Two genes were confirmed as differentially expressed between a set of ASC and normal adult samples by real-time RT-PCR. CONCLUSIONS: Differentially expressed genes in peripheral blood mononuclear cells between ASCs and normal adults were isolated by suppression subtractive hybridization, and included some new genes. Of the upregulated genes in ASCs, checkpoint suppressor I is associated with DNA damage-induced cell cycle arrest. Perforin I is associated with inflammation. The information about such alterations in gene expression could be useful for elucidating the mechanisms of ASC pathogenesis.展开更多
BACKGROUND The global outbreak of coronavirus disease 2019(COVID-19)leads to the development of accessible and cost-effective rapid antigen-detection tests(RATs),as quick and accurate diagnosis is crucial to curb the ...BACKGROUND The global outbreak of coronavirus disease 2019(COVID-19)leads to the development of accessible and cost-effective rapid antigen-detection tests(RATs),as quick and accurate diagnosis is crucial to curb the pandemic.AIM To evaluate the Humasis COVID-19 Ag Test(Humasis Co.,Ltd.,Gyeonggi-do,Republic of Korea)in the diagnosis of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2).METHODS This retrospective study was carried out at the Croatian Institute of Public Health and included patients with clinical symptoms of COVID-19 lasting no longer than 5 d prior to testing,whose nasopharyngeal swabs were primarily tested with RAT.Negative RAT samples underwent confirmatory real-time reverse transcription-polymerase chain reaction(RT-PCR).Diagnostic efficacy was determined compared to RT-PCR.The patients were divided into three age groups(<18,19-65,>65 years).Statistical analysis was performed with the significance level set at P<0.05.RESULTSIn total,2490 symptomatic patients were tested;953 samples were positive on RAT,and 1537 werenegative.All negative RAT samples were subjected to RT-PCR;266 samples were positive andmarked as false-negative results on RAT.The calculated negative predictive value as a measure ofRAT efficacy was 82.69%.The χ^(2) test and Kruskal-Wallis test showed a significant difference in theproportion of false negatives(P<0.001)and RT-PCR cycle(Ct)values for false-negative RATs(P=0.012)among the age groups.The young age group was significantly less likely to be falsenegative,whereas the false negatives from the elderly group experienced significantly lower Ctvalues than the other two age groups.CONCLUSIONEvaluated RAT demonstrated satisfactory performance with more reliable results in youngerpatients.Humasis COVID-19 Ag RAT is potentially a valuable tool in areas where access tomolecular methods is limited;however,RT-PCR remains a gold standard for SARS-CoV-2detection.展开更多
Objective: To investigate the expression of PTEN in endometrial carcinoma and its clinical significance. Methods: Reverse transcriptase-polymerase chain reaction and Western-blot methods were used to detect PTEN expre...Objective: To investigate the expression of PTEN in endometrial carcinoma and its clinical significance. Methods: Reverse transcriptase-polymerase chain reaction and Western-blot methods were used to detect PTEN expression in 28 cases of endometrial carcinoma. Results: mRNA and protein expression levels of PTEN in endometrial carcinomas were significantly lower than those in normal endometrium (P<0.01). Conclusion: PTEN may play an important role in the tumorigenesis of endometrial carcinoma.展开更多
Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was ...Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.展开更多
Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse t...Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-l, jmjdla, jmjd2c, and cyclin DI. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expression significantly decreased, whereas expression of neuronal markers, such as microtubule associated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.展开更多
Defining con-founders that affect the reliability of diagnostic tests for coronavirus disease 2019 is vital to breaking the chain of infection.The elderly population is a higher risk group for the emerging virus.Howev...Defining con-founders that affect the reliability of diagnostic tests for coronavirus disease 2019 is vital to breaking the chain of infection.The elderly population is a higher risk group for the emerging virus.However,gender seems to exert a critical role in modifying the infection risk among women owing to hormonal changes.The menopause transition is an exceptional period for older women where the protective and immunomodulatory effects of the estrogen hormone are lost.Accordingly,attention should be given to postmenopausal women since they will have an increased risk compared to their pre-menopausal peers.展开更多
In this study, the effect of ascorbic acid 2-phosphate (Asc2P) was tested on porcine and rat mature hepatocytes in vitro. a). Asc2P increased the porcine, but not rat, albumin secretion and mRNA expression. The enhanc...In this study, the effect of ascorbic acid 2-phosphate (Asc2P) was tested on porcine and rat mature hepatocytes in vitro. a). Asc2P increased the porcine, but not rat, albumin secretion and mRNA expression. The enhancing effect of Asc2P on porcine C/EBP alpha mRNA was observed in porcine mature hepatocytes. These data suggested that Asc2P played an important role in the regulation of porcine albumin mRNA level. b). The enhancing effect of Asc2P on ammonium metabolic activity was also observed in porcine, but not rat, mature hepatocytes. The porcine ornithine transcarbamylase (OTC) and arginase mRNAs were augmented by Asc2P, indicating that Asc2P had a direct effect on the urea cycle. c). The porcine collagen type I and type III mRNA, but not type XII mRNA, were detected as well, sugessting that Asc2P did not have the effect on the non-parenchymal hepatocytes to induce collagen type I and III mRNA expression. d). Our RT-PCR analysis demonstrated that the porcine hepatocytes expressed the sodium-ascorbate co-transporters SVCT1 and SVCT2, however, the intensities of porcine sodium-ascorbate co-transporters SVCT1 and SVCT2 bands were not changed markedly. These findings indicated that the Asc2P had no effect on SVCT1 and SVCT2 mRNA expression. e). The enhancing effect of Asc2P on porcine albumin mRNA was inhibited by staurosporine, a portein kinase inhibitor. We conclude that the enhanced albumin mRNA by Asc2P might be due to activation of tyrosine protein kinase and/or PKC and the Asc2P enhanced porcine albumin mRNA mainly at the transcriptional step.展开更多
Background Ewing's sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is often difficuh to distinguish from other small round cell tumors. The EWS-Ets gene fusions that result from chromosomal translocat...Background Ewing's sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is often difficuh to distinguish from other small round cell tumors. The EWS-Ets gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival materials, we evaluated the feasibility of detecting EWS-Ets including EWS-Flil and EWS-ERG fusion transcripts in paraffin-embedded tissues and its diagnostic value for detecting ES/pPNET. Methods Thirteen paraffin-embedded samples of ES/pPNETs were retrieved from archives. Thirteen cases of other tumors with small round cell features (including rhabdomyosarcoma, neuroblastoma, lymphoma, small ceil carcinoma, and desmoplastic small round cell tumor ) were used as negative controls. β-actin and β2- microglobulin were used as internal controls. A nested reverse transcriptase-polymerase chain reaction (RT- PCR)-based assay was performed to detect the EWS-Flil and EWS-ERG fusion transcripts. Results β-aetin and β2-mieroglobulin were detected in 10/13 and 13/13 ES/pPNETs, respectively. EWS- Flil fusion transcripts were detected in 11 of 13 (85%) ES/pPNETs. Three chimeric transcripts, all EWS-Flil, were detected in ES/pPNET samples. Among 11 EWS-Flil-positive cases, 7 eases had a type Ⅰ fusion transcript involving fusion of EWS exon 7 with Flil exon 6, 2 eases had a type Ⅱ fusion transcript involving EWS exon 7 with Flil exon 5, and 2 eases expressed fusion transcripts involving EWS exon 7 and Flil exon 8. Type Ⅰ EWS- Flil fusion predominated over other types. Fusion types could not be distinguished in the remaining 2 eases. Thirteen negative controls did not show detectable chimeric messages. There was a significant relationship between EWS-Flil fusion transcripts and CD99 expression. Conclusions Molecular detection of EWS-Flil fusion transcripts in formalin-fixed paraffin-embedded material by nested RT-PCR is feasible and is useful for the diagnosis and differential diagnosis of ES/pPNETs.展开更多
The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(c...The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.展开更多
基金Supported by grant from Fundamental Research Grant Scheme by Ministry of Higher Education(MoHE)600-IRMI/FRGS 5/3(101/2019).
文摘AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.
文摘Risk of clinically significant prostate adenocarcinoma (CAP) varies worldwide,although there is a uniform prevalence of latent disease. A hormone-responsive tissue,the prostate possesses the metabolizing capacity to biotransform a variety of environmental procarcinogens or endogenous hormones. Whether such metabolizing capacity or estrogen receptor (ER) status underlies these demographic differences in susceptibility to CaP remains unclear. With appropriate ethical permission,verified-benign tissues were obtained following transurethral resection of the prostate from a high-risk region (n = 12 UK-resident Caucasians) and a typically low-risk region (n = 14 India-resident Asians). Quantitative gene expression analysis was employed for cytochrome P450 (CYP)1B1,N-acetyltransferase (NAT)1,NAT2,catechol-O-methyl transferase ( COMT),sulfotransferase ( SULT) 1A1,ERα,ERβ and aromatase (CYP To quantify the presence or absence of CYP1B1,ERα or ERβ,and to identify ther in situ localization,immunohistochemistry was carried out. The two cohorts had reasonably well-matched serum levels of prostate-specific antigen or hormones. Expression levels for the candidate genes investigated were similar.However,clear differences in protein levels for CYP1B1 and ERβ were noted. Staining for CYP1B1 tended to be nuclear-associated in the basal glandular epithelial cells,and in UK-resident Caucasian tissues was present at a higher (P = 0.006) level compared with that from India-resident Asians. In contrast,a higher level of positive ERβ staining was noted in prostates from India-resident Asians. These study findings point to differences in metabolizing capacity and ER status in benign prostate tissues that might modulate susceptibility to the emergence of clinically significant CaP in demographically distinct populations.
基金funded by the National Natural Science Foundation of China (30960271 and 31160493)the doctor fund project of Ministry of Education of China(20111515110008)
文摘The sheep genome harbours approximately 20 copies of endogenous beta-retroviruses (enJSRVs), and circumstantial evidence suggests that enJSRVs might play a role in mammalian reproduction, particularly placental morphogenesis. This study was aimed to assess the expression of mRNAs of an enJSRV and its receptor, HYAL2, in the uterus and conceptuses of Mongolian ewes throughout gestation, using real-time reverse transcription polymerase chain reaction and in situ hybridization analysis. The results showed that enJSRV and HYAL2 mRNAs were found to be expressed throughout gestation in the endometrium, chorion, placenta, and conceptus. The enJSRV mRNA was most abundant in the placenta on day 90 of pregnancy, in the endometrium on day 30 and 50, and in the chorion on day 70 and 110. However, HYAL2 mRNA was most abundant in the endometrium on day 30. These differences were all significantly different from each other (P〈0.01). In situ hybridization showed that enJSRV and HYAL2 mRNAs were specifically expressed in endometrial luminal epithelium and glandular epithelium, trophoblastic giant binucleated cells (BNCs), endometrial caruncles, placental cotyledons, stroma, trophectoderm, as well as multinucleated syncytia of the placenta and blood vessel endothelial cells. Collectively, little is known about the molecular mechanisms by which trophoblastic differentiation and multinucleated syncytia formation are regulated by enJSRVs. However, the temporal and spatial distributions of enJSRV expression in the uterus and conceptus indicate that differentiation of BNCs and the formation of a multinucleated syncytiotrophoblast involve enJSRV and possibly its cellular receptor, HYAL2. Therefore, enJSRV and HYAL2 appear to play important roles in the female reproductive physiology in this breed of sheep.
文摘BACKGROUND: Interstitial collagenase has been considered as an essential enzyme for collagenolysis in liver fi-brosis, because type Ⅰ and Ⅲ collagens increase predominantly in liver fibrosis. The present study aimed to demonstrate the gene expression of matrix metalloproteinases-13 (MMP-13) in the progressive phases of ethanol induced experimental liver fibrosis in rats. METHODS: Thirty-four Sprague-Dawley rats were randomly divided into two groups. The experimental group (24 rats) was given ethanol (44% , 7 g/kg) every day and the control group (10) was given normal saline. Liver samples were harvested from experimental rats at 4, 12 and 24 weeks respectively. The kinetics of MMP-13 mRNA expression was assayed by semi-quantity reverse transcriptase-poly-merase chain reaction (RT-PCR). RESULTS: In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR (0.24±0.41). The gene expression of MMP-13 was increased in the liver of the rats treated with ethanol for 4 weeks (0. 62 ±0. 54), but it was not considered statistically significant (P >0.05). And the livers from 12-week-treated rats showed a marked mRNA expression(1.65 ±0.47, P <0. 01). Once fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39±0.25). CONCLUSION: MMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, and it was induced in a distinct time frame.
基金the Jawaharlal Nehru University(JNU) research fellowship sponsored by the Department of Biotechnology(DBT),Government of India
文摘Drought is one of the major abiotic stresses which adversely affect crop plants limiting growth and yield potential.Structural and functional characterization of drought stress-induced genes has contributed to a better understanding of how plants respond and adapt to the drought stress.In the present study,differential display technique was employed to study the gene expression of rice plants at the reproductive stage that were subjected to drought stress by withholding water,Pseudomonas fluorescens strain(Pf1) treated plants subjected for drought stress by withholding water and control(well-watered).Differentially expressed c DNAs of six genes(COX1,PKDP,b ZIP1,AP2-EREBP,Hsp20 and COC1) were identified,cloned and sequenced.Real-time q PCR analysis showed that all the six genes were upregulated in drought-stressed plants treated with Pf1.This revealed that the remarkable influence of Pf1 colonization leads to drought tolerance at the reproductive stage.These results showed that high levels of gene expression in plants lacking adequate water can be remarkably influenced by Pf1 colonization,which might be a key element for induced systemic tolerance by microbes.
文摘Summary: In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor β (TGF-β), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical methods were used for detection of TGF-β mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-β immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-β, and LEC could be affected by TGF-β through autocrine action.
基金supported by a grant from the Science and Technology Department of Zhejiang Province(No.2003C13015)
文摘BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adults clear virus from primary HBV infection, so the difference of gene expression between ASCs and normal adults determines the differential outcomes. To identify differentially expressed genes in ASCs compared to normal adults, we used suppression subtractive hybridization to compare gene expression. METHODS: cDNA subtracted libraries were constructed by suppression subtracted hybridization from peripheral blood monocytes of ASCs and normal adults, the subtracted clones were prescreened by dot blot hybridization, and the levels of genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: One hundred and two and 96 positive clones were acquired from the A-N and N-A libraries, respectively, and 14 and 11 clones were identified by dot blot hybridization in the A-N and N-A libraries. Two genes were confirmed as differentially expressed between a set of ASC and normal adult samples by real-time RT-PCR. CONCLUSIONS: Differentially expressed genes in peripheral blood mononuclear cells between ASCs and normal adults were isolated by suppression subtractive hybridization, and included some new genes. Of the upregulated genes in ASCs, checkpoint suppressor I is associated with DNA damage-induced cell cycle arrest. Perforin I is associated with inflammation. The information about such alterations in gene expression could be useful for elucidating the mechanisms of ASC pathogenesis.
文摘BACKGROUND The global outbreak of coronavirus disease 2019(COVID-19)leads to the development of accessible and cost-effective rapid antigen-detection tests(RATs),as quick and accurate diagnosis is crucial to curb the pandemic.AIM To evaluate the Humasis COVID-19 Ag Test(Humasis Co.,Ltd.,Gyeonggi-do,Republic of Korea)in the diagnosis of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2).METHODS This retrospective study was carried out at the Croatian Institute of Public Health and included patients with clinical symptoms of COVID-19 lasting no longer than 5 d prior to testing,whose nasopharyngeal swabs were primarily tested with RAT.Negative RAT samples underwent confirmatory real-time reverse transcription-polymerase chain reaction(RT-PCR).Diagnostic efficacy was determined compared to RT-PCR.The patients were divided into three age groups(<18,19-65,>65 years).Statistical analysis was performed with the significance level set at P<0.05.RESULTSIn total,2490 symptomatic patients were tested;953 samples were positive on RAT,and 1537 werenegative.All negative RAT samples were subjected to RT-PCR;266 samples were positive andmarked as false-negative results on RAT.The calculated negative predictive value as a measure ofRAT efficacy was 82.69%.The χ^(2) test and Kruskal-Wallis test showed a significant difference in theproportion of false negatives(P<0.001)and RT-PCR cycle(Ct)values for false-negative RATs(P=0.012)among the age groups.The young age group was significantly less likely to be falsenegative,whereas the false negatives from the elderly group experienced significantly lower Ctvalues than the other two age groups.CONCLUSIONEvaluated RAT demonstrated satisfactory performance with more reliable results in youngerpatients.Humasis COVID-19 Ag RAT is potentially a valuable tool in areas where access tomolecular methods is limited;however,RT-PCR remains a gold standard for SARS-CoV-2detection.
文摘Objective: To investigate the expression of PTEN in endometrial carcinoma and its clinical significance. Methods: Reverse transcriptase-polymerase chain reaction and Western-blot methods were used to detect PTEN expression in 28 cases of endometrial carcinoma. Results: mRNA and protein expression levels of PTEN in endometrial carcinomas were significantly lower than those in normal endometrium (P<0.01). Conclusion: PTEN may play an important role in the tumorigenesis of endometrial carcinoma.
基金This project was supported by the National Natural ScienceFoundation of China ( Serial No.3 8970 75 8)
文摘Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.
基金the research grant No. 1.1266 from International Centre for Science, High Technology and Environmental Sciences
文摘Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-l, jmjdla, jmjd2c, and cyclin DI. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expression significantly decreased, whereas expression of neuronal markers, such as microtubule associated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.
文摘Defining con-founders that affect the reliability of diagnostic tests for coronavirus disease 2019 is vital to breaking the chain of infection.The elderly population is a higher risk group for the emerging virus.However,gender seems to exert a critical role in modifying the infection risk among women owing to hormonal changes.The menopause transition is an exceptional period for older women where the protective and immunomodulatory effects of the estrogen hormone are lost.Accordingly,attention should be given to postmenopausal women since they will have an increased risk compared to their pre-menopausal peers.
文摘In this study, the effect of ascorbic acid 2-phosphate (Asc2P) was tested on porcine and rat mature hepatocytes in vitro. a). Asc2P increased the porcine, but not rat, albumin secretion and mRNA expression. The enhancing effect of Asc2P on porcine C/EBP alpha mRNA was observed in porcine mature hepatocytes. These data suggested that Asc2P played an important role in the regulation of porcine albumin mRNA level. b). The enhancing effect of Asc2P on ammonium metabolic activity was also observed in porcine, but not rat, mature hepatocytes. The porcine ornithine transcarbamylase (OTC) and arginase mRNAs were augmented by Asc2P, indicating that Asc2P had a direct effect on the urea cycle. c). The porcine collagen type I and type III mRNA, but not type XII mRNA, were detected as well, sugessting that Asc2P did not have the effect on the non-parenchymal hepatocytes to induce collagen type I and III mRNA expression. d). Our RT-PCR analysis demonstrated that the porcine hepatocytes expressed the sodium-ascorbate co-transporters SVCT1 and SVCT2, however, the intensities of porcine sodium-ascorbate co-transporters SVCT1 and SVCT2 bands were not changed markedly. These findings indicated that the Asc2P had no effect on SVCT1 and SVCT2 mRNA expression. e). The enhancing effect of Asc2P on porcine albumin mRNA was inhibited by staurosporine, a portein kinase inhibitor. We conclude that the enhanced albumin mRNA by Asc2P might be due to activation of tyrosine protein kinase and/or PKC and the Asc2P enhanced porcine albumin mRNA mainly at the transcriptional step.
文摘Background Ewing's sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is often difficuh to distinguish from other small round cell tumors. The EWS-Ets gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival materials, we evaluated the feasibility of detecting EWS-Ets including EWS-Flil and EWS-ERG fusion transcripts in paraffin-embedded tissues and its diagnostic value for detecting ES/pPNET. Methods Thirteen paraffin-embedded samples of ES/pPNETs were retrieved from archives. Thirteen cases of other tumors with small round cell features (including rhabdomyosarcoma, neuroblastoma, lymphoma, small ceil carcinoma, and desmoplastic small round cell tumor ) were used as negative controls. β-actin and β2- microglobulin were used as internal controls. A nested reverse transcriptase-polymerase chain reaction (RT- PCR)-based assay was performed to detect the EWS-Flil and EWS-ERG fusion transcripts. Results β-aetin and β2-mieroglobulin were detected in 10/13 and 13/13 ES/pPNETs, respectively. EWS- Flil fusion transcripts were detected in 11 of 13 (85%) ES/pPNETs. Three chimeric transcripts, all EWS-Flil, were detected in ES/pPNET samples. Among 11 EWS-Flil-positive cases, 7 eases had a type Ⅰ fusion transcript involving fusion of EWS exon 7 with Flil exon 6, 2 eases had a type Ⅱ fusion transcript involving EWS exon 7 with Flil exon 5, and 2 eases expressed fusion transcripts involving EWS exon 7 and Flil exon 8. Type Ⅰ EWS- Flil fusion predominated over other types. Fusion types could not be distinguished in the remaining 2 eases. Thirteen negative controls did not show detectable chimeric messages. There was a significant relationship between EWS-Flil fusion transcripts and CD99 expression. Conclusions Molecular detection of EWS-Flil fusion transcripts in formalin-fixed paraffin-embedded material by nested RT-PCR is feasible and is useful for the diagnosis and differential diagnosis of ES/pPNETs.
文摘The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.