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Blanda, Valeria, Rosalia D’Agostino, Elisabetta Giudice, Kety Randazzo, Francesco La Russa, Sara Villari, Stefano Vullo, and Alessandra Torina. "New Real-Time PCRs to Differentiate Rickettsia spp. and Rickettsia conorii." Molecules 25, no. 19 (September 27, 2020): 4431. http://dx.doi.org/10.3390/molecules25194431.
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Rickettsia species are an important cause of emerging infectious diseases in people and animals, and rickettsiosis is one of the oldest known vector-borne diseases. Laboratory diagnosis of Rickettsia is complex and time-consuming. This study was aimed at developing two quantitative real-time PCRs targeting ompB and ompA genes for the detection, respectively, of Rickettsia spp. and R. conorii DNA. Primers were designed following an analysis of Rickettsia gene sequences. The assays were optimized using SYBR Green and TaqMan methods and tested for sensitivity and specificity. This study allowed the development of powerful diagnostic methods, able to detect and quantify Rickettsia spp. DNA and differentiate R. conorii species.
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Joly, Philippe, Pierre-Alain Falconnet, Janine André, Nicole Weill, Monique Reyrolle, François Vandenesch, Max Maurin, Jerome Etienne, and Sophie Jarraud. "Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation." Applied and Environmental Microbiology 72, no. 4 (April 2006): 2801–8. http://dx.doi.org/10.1128/aem.72.4.2801-2808.2006.
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ABSTRACT Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >103 CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.
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Ruszova, E., M. Chmelarova, M. Senkerikova, and S. Stefackova. "Duplex Methylation-Specific Semi-Quantitative Real-Time PCR for Cost-Effective & Time-Efficient Diagnostic Screening of Chromosome 15 and 14 Imprinted Regions." Acta Medica Martiniana 15, no. 3 (December 1, 2015): 5–12. http://dx.doi.org/10.1515/acm-2015-0012.
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AbstractPurpose: Our goal was to develop two-tier strategy based onin house-designed methylation specific-duplex polymerase chain reactions (MS-PCRs) that could serve as a relatively simple, cost effective, time efficient approach for molecular screening of imprinted regions on chromosomes 15 and 14.Patients and methods: Patients were referred to examination during infancy due to hypotonia and motor development delay. Duplex MS-PCRs were performed that enabled detection of methylated/unmethylated DNA inNDNandMEG3CpG islands via plurality of detection channels on PCR instrument Rotor Gene 6000.Results and discussion: Both, copy number variations as well as methylation changes, were revealed by ourin house-designed methodology by focusingNDNgene. No imprinting aberrations were yet discovered inMEG3gene. Clinical features of the patients were compared. In agree with literature no typical facial features were observed in PWS patient with imprinting defect and AS UPD patient seems to have a relatively better development and language ability in comparison to deletional form of the disease.Conclusion: In conclusion we were able to establish new, throughput and robust diagnostic approach to PWS/AS.
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Papp, Stefanie, Jessica Rauch, Svenja Kuehl, Ulricke Richardt, Christian Keller, and Anke Osterloh. "Comparative evaluation of two Rickettsia typhi-specific quantitative real-time PCRs for research and diagnostic purposes." Medical Microbiology and Immunology 206, no. 1 (October 1, 2016): 41–51. http://dx.doi.org/10.1007/s00430-016-0480-z.
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Lopotova, Tereza, Jana Moravcova, Vaclava Polivkova, Jaroslav Polak, Jiri Schwarz, Hana Klamova, and Katerina Machova Polakova. "Expression of Four Major WT1 Splicing Variants in AML and CML: Development of Quantitative Real-Time PCRs and Preliminary Results in Patient Samples." Blood 114, no. 22 (November 20, 2009): 1631. http://dx.doi.org/10.1182/blood.v114.22.1631.1631.
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Abstract Abstract 1631 Poster Board I-657 WT1 expression has proved to be of prognostic significance in AML, MDS and according to our unpublished data also in CML patients. There are four major WT1 isoforms with distinct functions in cells. Splicing variants encoding the WT1 isoforms differ from each other by the presence or absence of both the exon 5 and KTS region in the exon 9 of WT1 gene. It was shown that exon 5 containing isoforms appear to cause cell cycle arrest. Therefore, differences in WT1 splicing variant ratios might be associated with prognosis and response to therapy. All currently used primer/probe systems quantify all major splicing variants as expression of total WT1. The aim of our study was to develop specific quantitative real-time PCRs (qRT-PCRs) for quantification of the four major WT1 splicing variants (+5+KTS; +5-KTS; -5+KTS; -5-KTS) at mRNA level and to apply these techniques for initial screening of WT1 splicing variants expression in AML and CML patients. We designed several sequence variants of two forward (specific for +/-5) and two reverse primers (specific for +/-KTS) and one common TaqMan probe. After in silico analyses, selected primers were subjected for testing with plasmids each carrying target sequence of the splicing variant. The qRT-PCRs were performed on RotorGene (Corbett Research). GUS was used as housekeeping gene; primers, probes and protocols were adopted from Gabert et al. (Leukemia 2003, 17:2318). Absolute quantification was applied for data evaluation (reported in percentage). Most of these tests focused on qRT-PCR specificity. The main test of quantification utility and specificity was performed by using 25 plasmid mixtures that simulate different ratios of particular variants (copies of each variant were in five different ratios to the remaining three variants; from 10/100000 to 100000/10 copies). Finally, total leukocytes from 45 AML and 13 CML patients at diagnosis were analysed to test system utility on real samples. The splicing ratio stability during the time was checked, because there were some specimens of peripheral blood not processed immediately but preserved at 4°C overnight. Our results showed high specificity and utility of all four qRT-PCRs. We were able to quantify up to 100 copies of each variant. Even in case that one of the variants was in minority compared to others in the plasmid mixture, expected number of plasmid copies was assessed by the qRT-PCR. Splicing variant ratio was found to be conserved, when the blood was preserved overnight in refrigerator. Observed differences fall within the ranges of the technical variability of the four qRT-PCRs (12-50%). In general, our preliminary data in patient samples showed the +5/-KTS variant as the most abundant in patients with CML and AML. Expression of the +5/-KTS and -5/+KTS highly varied among AML patients within the same FAB subtype. Low level of +5/+KTS variant (median 18% from total WT1) distinguished the high risk group patients (cytogenetic-based risk stratification) from the remaining groups (median 30% from total WT1). Our data also indicate that +KTS/-KTS variant expression levels independent of the presence of exon 5 may be of importance. While in low and intermediate risk groups +KTS and –KTS variants were expressed without significant differences, in the high risk group of patients the expression of –KTS variants was significantly higher. At the same time, the high risk group showed significantly decreased +5 variants levels (independent of the presence of KTS sequence). In conclusion, we have developed the specific qRT-PCRs detecting the four WT1 splicing variants on mRNA level. Further research is ongoing to confirm the aforementioned significance of different expression of the particular WT1 isoforms. Supported by MZOUHKT2005. Disclosures No relevant conflicts of interest to declare.
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Maksimov, Pavlo, Hannes Bergmann, Marion Wassermann, Thomas Romig, Bruno Gottstein, Adriano Casulli, and Franz J. Conraths. "Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs." Pathogens 9, no. 10 (September 26, 2020): 791. http://dx.doi.org/10.3390/pathogens9100791.
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Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.
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Wall, Steven J., and Dylan R. Edwards. "Quantitative Reverse Transcription–Polymerase Chain Reaction (RT-PCR): A Comparison of Primer-Dropping, Competitive, and Real-Time RT-PCRs." Analytical Biochemistry 300, no. 2 (January 2002): 269–73. http://dx.doi.org/10.1006/abio.2001.5458.
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Gautam, Rashi, Slavica Mijatovic-Rustempasic, Mathew D. Esona, Ka Ian Tam, Osbourne Quaye, and Michael D. Bowen. "One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples." PeerJ 4 (January 11, 2016): e1560. http://dx.doi.org/10.7717/peerj.1560.
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Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.
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Wen, Jing, Hong Yang, Kongjia Luo, Yi Hu, Xu Zhang, Geng Wang, Yuping Chen, et al. "A three-gene expression signature model to predict neo-chemoradiotherapy response of esophageal squamous cell carcinomas." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e15135-e15135. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e15135.
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e15135 Background: Preoperative chemoradiotherapy (CRT) followed by surgery has been proved to improve survival in comparison with surgery alone. However, the outcomes of CRT are heterogeneous, and no clinical or pathological method could prediction CRT response. In this study, we aim to identify mRNA markers for ESCC CRT-response prediction. Methods: Gene expression analyses were performed on pretreatment cancer biopsies from 28 ESCCs who received neoadjuvant CRT and surgery. Surgical specimens were assessed for the pathological response to CRT. The identified differentially expressed genes were validated by real-time quantitative polymerase chain reaction (qPCR), based on which a classifying model was built up by Fisher’s linear discriminant analysis. The predictive power of this model was further assessed in another set of 32 ESCCs. Results: The profiling of the 28 ESCCs identified 10 differentially expressed genes with more than 2-fold changes between pathological complete responsers (pCRs) and less than pCRs (<pCRs), among which 6 genes (LIMCH1, SDPR, Clorf226, SLC9A9, GSTM3, and IGSF10) were down-regulated and 4 genes (MMP9, MMP1, MMP12 and OASL) up-regulated in pCRs versus <pCRs. A prediction model based on qPCR values of 3 genes was built up, Y=-10.388 - 0.343 × MMP1 + 0.642 × LIMCH1 + 0.921 × Clorf226 with a cut-off value of 0.607. It provided a predictive accuracy of 85.7% with leave-one-out cross-validation. Further, the predictive power of this model was validated in another set of 32 ESCCs, among which a predictive accuracy of 81.3% was achieved. Conclusions: The combination of three genes by qPCR identified by microarrays in our study provides possibility for ESCC CRT prediction, which will facilitate individualization of ESCC treatment. Further perspective validation in larger independent cohorts is warranted to fully assess the predictive power of this prediction model.
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Skubic, Lucijan, Lea Hošnjak, Vesna Breznik, Kristina Fujs Komloš, Boštjan Luzar, and Mario Poljak. "An Improved Protocol for Comprehensive Etiological Characterization of Skin Warts and Determining Causative Human Papillomavirus Types in 128 Histologically Confirmed Common Warts." Viruses 14, no. 10 (October 15, 2022): 2266. http://dx.doi.org/10.3390/v14102266.
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Human papillomaviruses (HPVs) are etiologically associated with various benign and malignant neoplasms of cutaneous and mucosal epithelia. We describe an improved diagnostic protocol for comprehensive characterization of causative HPV types in common warts, in which broad-spectrum PCRs followed by Sanger sequencing, two previously described and seven newly developed type-specific quantitative real-time PCRs (qPCRs) coupled with the human beta-globin qPCR were used for: (i) diagnosis of HPV infection in warts; (ii) estimation of cellular viral loads of all HPV types detected; and (iii) determination of their etiological role in 128 histologically confirmed fresh-frozen common wart tissue samples. A total of 12 different causative HPV types were determined in 122/126 (96.8%) HPV-positive warts, with HPV27 being most prevalent (27.0%), followed by HPV57 (26.2%), HPV4 (15.1%), HPV2 (13.5%), and HPV65 (7.9%). The cellular viral loads of HPV4 and HPV65 were estimated for the first time in common warts and were significantly higher than the viral loads of HPV2, HPV27, and HPV57. In addition, we showed for the first time that HPV65 is etiologically associated with the development of common warts in significantly older patients than HPV27 and HPV57, whereas HPV4-induced warts were significantly smaller than warts caused by HPV2, HPV27, HPV57, and HPV65.
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SCHNIDER, A., G. OVERESCH, B. M. KORCZAK, and P. KUHNERT. "Comparison of Real-Time PCR Assays for Detection, Quantification, and Differentiation of Campylobacter jejuni and Campylobacter coli in Broiler Neck Skin Samples†." Journal of Food Protection 73, no. 6 (June 1, 2010): 1057–63. http://dx.doi.org/10.4315/0362-028x-73.6.1057.
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We tested the use of multiplex real-time PCR for detection and quantification of Campylobacter jejuni and Campylobacter coli on broiler carcass neck skin samples collected during 2008 from slaughterhouses in Switzerland. Results from an established TaqMan assay based on two different targets (hipO and ceuE for C. jejuni and C. coli, respectively) were corroborated with data from a newly developed assay based on a single-nucleotide polymorphism in the fusA gene, which allows differentiation between C. jejuni and C. coli. Both multiplex real-time PCRs were applied simultaneously for direct detection, differentiation, and quantification of Campylobacter from 351 neck skin samples and compared with culture methods. There was good correlation in detection and enumeration between real-time PCR results and quantitative culture, with real-time PCR being more sensitive. Overall, 251 (71.5%) of the samples were PCR positive for Campylobacter, with 211 (60.1%) in the hipO–ceuE assays, 244 (69.5%) in the fusA assay, and 204 (58.1%) of them being positive in both PCR assays. Thus, the fusA assay was similarly sensitive to the enrichment culture (72.4% positive); however, it is faster and allows for quantification. In addition, real-time PCR allowed for species differentiation; roughly 60% of positive samples contained C. jejuni, less than 10% C. coli, and more than 30% contained both species. Real-time PCR proved to be a suitable method for direct detection, quantification, and differentiation of Campylobacter from carcasses, and could permit time-efficient surveillance of these zoonotic agents.
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Escaffre, O., M. Queguiner, and N. Eterradossi. "Development and validation of four real-time quantitative RT-PCRs specific for the positive or negative strands of a bisegmented dsRNA viral genome." Journal of Virological Methods 170, no. 1-2 (December 2010): 1–8. http://dx.doi.org/10.1016/j.jviromet.2010.07.009.
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Mengelle, C., J. M. Mansuy, K. Sauné, C. Barthe, J. Boineau, and J. Izopet. "A new highly automated extraction system for quantitative real-time PCRs from whole blood samples: Routine monitoring of opportunistic infections in immunosuppressed patients." Journal of Clinical Virology 53, no. 4 (April 2012): 314–19. http://dx.doi.org/10.1016/j.jcv.2012.01.002.
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Hong, Kyeong Man, Hazim Najjar, Mary Hawley, and Richard D. Press. "Quantitative Real-Time PCR with Automated Sample Preparation for Diagnosis and Monitoring of Cytomegalovirus Infection in Bone Marrow Transplant Patients." Clinical Chemistry 50, no. 5 (May 1, 2004): 846–56. http://dx.doi.org/10.1373/clinchem.2003.026484.
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Abstract Background: In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. Methods: We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The β-globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. Results: The assay was linear (R = 0.999) from a lower detection limit of 125 copies/mL to 5 × 109 copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1–4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R = 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. Conclusions: The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.
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Kitzmiller, Kathryn J., Yan Yang, Yee Ling Wu, Bi Zhou, Erwin K. Chung, and C. Yung Yu. "Determining the copy number variation (CNV) of human Complement Factor H Related Genes: from Southern blots to Real-time PCRs (136.30)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 136.30. http://dx.doi.org/10.4049/jimmunol.182.supp.136.30.
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Abstract Current applications for detecting the CNV of Complement Factor H-Related Genes 3 and 1, CFHR3 and CFHR1, such as Multiplex Ligation-Dependent Probe Amplification, MLPA, rely on the presence of single nucleotide polymorphisms that associate with but do not necessarily reflect the actual physical variation of gene copy numbers. A 76-kb "deletion" of genomic region for CFHR3-CFHR1was detectable using long range-mapping of PmeI-digested DNA resolved by Pulsed-Field Electrophoresis using a CFHR1 cDNA probe. The deletion was further characterized by Southern blots of genomic DNA digested by TaqI using CFHR2 cDNA probe, or PshAI-PvuII using CFH exons 21-22 probe. Thus, we developed quantitative Real-time PCR (RT-PCR) assays to detect the deleted region using 36 samples of genomic DNA characterized by Southern blot for CNV of CFHR3-CFHR1. Three RT-PCR assays were designed to measure a common region between CFHR3 and CFHR4, and CFHR1 and CFHR2, as well as intergenic region between CFHR3 and CFHR1using the standard curve method and obtaining the ratio between the target gene(s) and endogenous control. The 3 RT-PCR assays were able to correctly identify the CNV of CFHR3-R1 in 35/36 samples, for 97.5% accuracy and 100% sensitivity. This method will enable further investigation of CFHR3-R1CNVs in Age-Related Macular Degeneration, atypical Hemolytic Anemia and Systemic Lupus Erythematosus.
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Kuribara, Hideo, Yoichiro Shindo, Takeshi Matsuoka, Ken Takubo, Satoshi Futo, Nobutaro Aoki, Takashi Hirao, et al. "Novel Reference Molecules for Quantitation of Genetically Modified Maize and Soybean." Journal of AOAC INTERNATIONAL 85, no. 5 (September 1, 2002): 1077–89. http://dx.doi.org/10.1093/jaoac/85.5.1077.
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Abstract New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye, labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0%level, the bias (mean–true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.
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Bohmova, Jana, Marek Lubusky, Iva Holuskova, Martina Studnickova, Romana Kratochvilova, Eva Krejcirikova, Veronika Durdova, et al. "Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma." Diagnostics 10, no. 8 (August 5, 2020): 564. http://dx.doi.org/10.3390/diagnostics10080564.
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Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.
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van den Munckhof, Ellen H. A., Rosalie L. van Sitter, Kim E. Boers, Ronald F. Lamont, René te Witt, Saskia le Cessie, Cornelis W. Knetsch, et al. "Comparison of Amsel criteria, Nugent score, culture and two CE-IVD marked quantitative real-time PCRs with microbiota analysis for the diagnosis of bacterial vaginosis." European Journal of Clinical Microbiology & Infectious Diseases 38, no. 5 (March 22, 2019): 959–66. http://dx.doi.org/10.1007/s10096-019-03538-7.
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PATON, Michael G., S. H. P. Parakrama KARUNARATNE, Elsa GIAKOUMAKI, Neil ROBERTS, and Janet HEMINGWAY. "Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes." Biochemical Journal 346, no. 1 (February 8, 2000): 17–24. http://dx.doi.org/10.1042/bj3460017.
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The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estα21 and estβ21 genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estα21 and estβ21 are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estβ21 gene from this amplicon has greater transcription than estα21 in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.
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M. Zakaria, Amira. "Comparative Assessment of Sensitivity and Specificity of Rose Bengal Test and Modified In-house ELISA by using IS711 TaqMan Real Time PCR Assay as a Gold Standard for the Diagnosis of Bovine Brucellosis." Biomedical and Pharmacology Journal 11, no. 2 (June 21, 2018): 951–57. http://dx.doi.org/10.13005/bpj/1453.
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Serological approaches such as Rose Bengal Test (RBT) and enzyme linked immunosorbent assay (ELISA) are common tests, however they are generally not sensitive or specific enough for diagnosis of Brucella because of cross-reactivity with different bacterial antigens. The work objected to evaluate the sensitivity and specificity of rose Bengal and modified in-house ELISA using IS711 real time PCR as a gold test to detect Brucella in calves sera. Two hundred and thirty (n=230) blood samples were collected from (2-3) years asymptomatic male calves in two Egyptian abattoirs. Rose Bengal test (RBT) and modified in-house ELISA were applied to determine the seroprevalence of brucellosis in abattoirs animals while quantitative Taqman real-time PCRs (RT-PCR) were implemented for the identification of Brucella genus. The overall prevalence of brucellosis was (53.9 %), (75.2 %) and (79.1 %) as determined by ELISA,RBT and RT- PCR assays respectively. Regarding statistical analysis of the reported data and considering real time PCR the gold standard, the RBT recorded a sensitivity of (79.12%) and a specificity of (39.58 %) with an accuracy of (70.87%). While (83.24%) was reported as positive predictive value and (33.33 %) as a negative predictive value. The sensitivity and specificity of ELISA were (55.49%) and (52.08 %) respectively while the accuracy was (54.78%). Positive predictive value and negative predictive value for ELISA were determined as (81.45%) and (23.58 %) respectively. RBT can be routinely used for diagnosis of Brucella as cost effective , more sensitive and accurate test than ELISA However, real time PCR is highly recommend as gold test for identification and differentiation of bovine brucellosis.
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Čermáková, Eliška, Kamila Zdeňková, Kateřina Demnerová, and Jaroslava Ovesná. "Comparison of methods to extract PCR-amplifiable DNA from fruit, herbal and black teas." Czech Journal of Food Sciences 39, No. 5 (October 14, 2021): 410–17. http://dx.doi.org/10.17221/24/2021-cjfs.
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The success of polymerase chain reaction (PCR) assay depends on template deoxyribonucleic acid (DNA) being sufficient with respect to both quantity and quality. Some biological materials contain compounds which inhibit the functioning of DNA polymerase and thus need to be removed as part of the DNA extraction procedure. The aim of the present experiments was to optimise the process of DNA isolation from various types of black, fruit and herbal teas. A comparison was made between two cetyltrimethylammonium bromide (CTAB)-based protocols and two commercially available DNA purification kits. The yield and integrity of the extracted DNA were monitored both spectrophotometrically and using agarose gel electrophoresis. The presence/absence of inhibitors in the DNA preparations was checked by running quantitative real-time PCRs. The optimal protocol was deemed to be the CTAB method described in ISO 21571:2005, so this method is recommended for the routine sample analysis of tea products.
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Jakkul, Wallop, Kittipong Chaisiri, Naowarat Saralamba, Yanin Limpanont, Sirilak Dusitsittipon, Vachirapong Charoennitiwat, Abigail Hui En Chan, and Urusa Thaenkham. "Newly developed SYBR Green-based quantitative real-time PCRs revealed coinfection evidence of Angiostrongylus cantonensis and A. malaysiensis in Achatina fulica existing in Bangkok Metropolitan, Thailand." Food and Waterborne Parasitology 23 (June 2021): e00119. http://dx.doi.org/10.1016/j.fawpar.2021.e00119.
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Zhang, Huan, and Senjie Lin. "Development of a cob-18S rRNA Gene Real-Time PCR Assay for Quantifying Pfiesteria shumwayae in the Natural Environment." Applied and Environmental Microbiology 71, no. 11 (November 2005): 7053–63. http://dx.doi.org/10.1128/aem.71.11.7053-7063.2005.
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ABSTRACT Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5′-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml−1), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).
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Delghandi, Mohammad Reza, Mansour El-Matbouli, and Simon Menanteau-Ledouble. "Renibacterium salmoninarum—The Causative Agent of Bacterial Kidney Disease in Salmonid Fish." Pathogens 9, no. 10 (October 15, 2020): 845. http://dx.doi.org/10.3390/pathogens9100845.
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Renibacterium salmoninarum is one of the oldest known bacterial pathogens of fish. This Gram-positive bacterium is the causative agent of bacterial kidney disease, a chronic infection that is mostly known to infect salmonid fish at low temperatures. Externally, infected fish can display exophthalmia as well as blebs on the skin and ulcerations alongside haemorrhages at the base of the fins and alongside the lateral line. Internally, the kidney, heart, spleen and liver can show signs of swelling. Granulomas can be seen on various internal organs, as can haemorrhages, and the organs can be covered with a false membrane. Ascites can also accumulate in the abdominal cavity. The bacterium is generally cultivated on specialized media such as kidney disease medium-1 (KDM-1), KDM-2 and selective kidney disease medium (SKDM), and a diagnostic is performed using molecular tools such as PCRs or real-time quantitative PCRs (RT-qPCRs). Several virulence mechanisms have been identified in R. salmoninarum, in particular the protein p57 that is known to play a role in both agglutination and immunosuppression of the host’s defense mechanisms. Control of the disease is difficult; the presence of asymptomatic carriers complicates the eradication of the disease, as does the ability of the bacterium to gain entrance inside the eggs. Bacterin-killed vaccines have proven to be of doubtful efficacy in controlling the disease, and even more recent application of a virulent environmental relative of R. salmoninarum is of limited efficacy. Treatment by antibiotics such as erythromycin, azithromycin and enrofloxacin can be effective but it is slow and requires prolonged treatment. Moreover, antibiotic-resistant strains have been reported. Despite being known for a long time, there is still much to be discovered about R. salmoninarum, notably regarding its virulence mechanisms and its vaccine potential. Consequently, these gaps in knowledge continue to hinder control of this bacterial disease in aquaculture settings.
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Li, Wen-han, Yong-chun Song, Hao Zhang, Zhang-jian Zhou, Xin Xie, Qing-nuo Zeng, Kun Guo, Ting Wang, Peng Xia, and Dong-min Chang. "Decreased Expression of Hsa_circ_00001649 in Gastric Cancer and Its Clinical Significance." Disease Markers 2017 (2017): 1–6. http://dx.doi.org/10.1155/2017/4587698.
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Background. It has been reported that circRNAs are differentially expressed in a wide range of cancers and could be used as a new biomarker for diagnosis. However, the correlation between circRNAs and gastric cancer (GC) it is still unclear. Materials and Methods. In this study, by using real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs), we detected the expression level of hsa_circ_0001649 in tissue and serum samples from GC patients. Results. We found that hsa_circ_0001649 expression was significantly downregulated in GC tissue compared with their paired paracancerous histological normal tissues (PCHNTs) (P<0.01). We next analyzed the expression level of hsa_circ_0001649 in serum samples between preoperative and postoperative GC patients. We found that its level in serum was significantly upregulated after surgery (P<0.01). The area under the receiver operating characteristic (ROC) curve was 0.834. Moreover, the expression level of hsa_circ_0001649 was significantly correlated with pathological differentiation (P=0.039). Conclusion. Our test suggested that hsa_circ_0001649 was significantly downregulated in GC and may become a novel potential biomarker in the diagnosis of GC.
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Taskin, Bilgin, Ayse Gul Gozen, and Metin Duran. "Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification." Applied and Environmental Microbiology 77, no. 13 (May 20, 2011): 4329–35. http://dx.doi.org/10.1128/aem.02895-10.
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ABSTRACTQuantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed withEscherichia coliATCC 25922 as the model organism and theuidAgene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1.
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Choi, Yeon-Jae, Sun-Ho Kim, Min-Jeong Gu, Han-Na Choe, Dong-Un Kim, Sang-Bum Cho, Su-Ki Kim, Che-Ok Jeon, Gui-Seok Bae, and Sang-Seok Lee. "Quantitative Real-time PCR using Lactobacilli as Livestock Probiotics." Journal of Life Science 20, no. 12 (December 30, 2010): 1896–901. http://dx.doi.org/10.5352/jls.2010.20.12.1896.
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Choe, Myeong-Eun, In-Jung Lee, and Jae-Ho Shin. "Assessment of Korean Paddy Soil Microbial Community Structure by Use of Quantitative Real-time PCR Assays." Korean Journal of Environmental Agriculture 30, no. 4 (December 31, 2011): 367–76. http://dx.doi.org/10.5338/kjea.2011.30.4.367.
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Zhou, Zhike, Jun Hou, and Qinghua Li. "Artesunate attenuates traumatic brain injury-induced impairments in rats." Translational Neuroscience 11, no. 1 (September 9, 2020): 309–18. http://dx.doi.org/10.1515/tnsci-2020-0136.
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AbstractBackgroundBlood–brain barrier (BBB) dysfunction and neuroinflammation induced by traumatic brain injuries (TBIs) cause a succession of secondary brain damage events and finally lead to a massive and progressive cerebral neuronal destruction. Artesunate, a semisynthetic artemisinin derivative, is a potential candidate for the management of cerebral damage induced by TBI due to its protective function to BBB and cerebral neurons.MethodsTo demonstrate the effect of artesunate to TBI-induced BBB dysfunction and neural damage, TBI rat model was constructed by cortical impact injury. Behavioral experiments were used to estimate the impact of the combined treatment on rats. Western blotting was performed to demonstrate the protein levels in the brain tissues of rats. Quantitative real-time PCRs were utilized to investigate the alteration in the expression of various RNA levels. The chemokine levels were estimated by ELISA.ResultsArtesunate treatment attenuated the impact caused by TBI on rat brain and improved the long-term neurological recover. Artesunate treatment protected the integrity of BBB and inhibited neuroinflammation. Artesunate treatment promoted the phosphorylation of Akt and inhibited the phosphorylation of glycogen synthase kinase (GSK)-3β in TBI rat model.ConclusionArtesunate protected rats from TBI-induced impairments of BBB and improved longer-term neurological outcomes.
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Barber, Robert D., Dan W. Harmer, Robert A. Coleman, and Brian J. Clark. "GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues." Physiological Genomics 21, no. 3 (May 11, 2005): 389–95. http://dx.doi.org/10.1152/physiolgenomics.00025.2005.
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Quantitative gene expression data are often normalized to the expression levels of control or so-called “housekeeping” genes. An inherent assumption in the use of housekeeping genes is that expression of the genes remains constant in the cells or tissues under investigation. Although exceptions to this assumption are well documented, housekeeping genes are of value in fully characterized systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. To investigate the value of GAPDH as a housekeeping gene in human tissues, the expression of GAPDH mRNA was measured in a panel of 72 different pathologically normal human tissue types. Measurements were obtained from 371,088 multiplexed, quantitative real-time RT-PCRs with specific target genes. Significant differences in the expression levels of GAPDH mRNA were observed between tissue types and between donors of the same tissue. A 15-fold difference in GAPDH mRNA copy numbers was observed between the highest and lowest expressing tissue types, skeletal muscle and breast, respectively. No specific effect of either age or gender was observed on GAPDH mRNA expression. These data provide an extensive analysis of GAPDH mRNA expression in human tissues and confirm previous reports of the marked variability of GAPDH expression between tissue types. These data establish comparative levels of expression and can be used to add value to gene expression data in which GAPDH is used as the internal control.
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Jeong, Hye Mi, and Kwang Yup Kim. "Quantitative Analysis of Feline Calicivirus Inactivation using Real-time RT-PCR." Journal of Food Hygiene and Safety 29, no. 1 (March 30, 2014): 31–39. http://dx.doi.org/10.13103/jfhs.2014.29.1.031.
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Rogucki, Mariusz, Iwona Sidorkiewicz, Magdalena Niemira, Janusz Bogdan Dzięcioł, Angelika Buczyńska, Agnieszka Adamska, Katarzyna Siewko, et al. "Expression Profile and Diagnostic Significance of MicroRNAs in Papillary Thyroid Cancer." Cancers 14, no. 11 (May 28, 2022): 2679. http://dx.doi.org/10.3390/cancers14112679.
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The incidence of papillary thyroid cancer (PTC) has increased in recent years. To improve the diagnostic management of PTC, we propose the use of microRNAs (miRNAs) as a biomarker. Our aim in this study was to evaluate the miRNA expression pattern in PTC using NanoString technology. We identified ten miRNAs deregulated in PTC compared with reference tissue: miR-146b-5p, miR-221-3p, miR-221-5p, miR-34-5p, miR-551b-3p, miR-152-3p, miR-15a-5p, miR-31-5p, and miR-7-5p (FDR < 0.05; |fold change (FC)| ≥ 1.5). The gene ontology (GO) analysis of differentially expressed miRNA (DEM) target genes identified the predominant involvement of epidermal growth factor receptor (EGFR), tyrosine kinase inhibitor resistance, and pathways in cancer in PTC. The highest area under the receiver operating characteristic (ROC) curve (AUC) for DEMs was found for miR-146-5p (AUC = 0.770) expression, indicating possible clinical applicability in PTC diagnosis. The combination of four miRNAs (miR-152-3p, miR-221-3p, miR-551b-3p, and miR-7-5p) showed an AUC of 0.841. Validation by real-time quantitative polymerase chain reactions (qRT-PCRs) confirmed our findings. The introduction of an miRNA diagnostic panel based on the results of our study may help to improve therapeutic decision making for questionable cases. The use of miRNAs as biomarkers of PTC may become an aspect of personalized medicine.
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Nadal, Anna, Teresa Esteve, and Maria Pla. "Multiplex Polymerase Chain Reaction-Capillary Gel Electrophoresis: A Promising Tool for GMO ScreeningAssay for Simultaneous Detection of Five Genetically Modified Cotton Events and Species." Journal of AOAC INTERNATIONAL 92, no. 3 (May 1, 2009): 765–72. http://dx.doi.org/10.1093/jaoac/92.3.765.
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Abstract A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7 of false classification rate), with limit of detection values of 0.1 for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.
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Lee, Dae-Weon. "Detection of a Microsporidium, Nosema ceranae, from Field Population of the Bumblebee, Bombus terrestris, via Quantitative Real-Time PCR." Korean Journal of Microbiology 49, no. 3 (September 30, 2013): 270–74. http://dx.doi.org/10.7845/kjm.2013.3052.
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Gendelman, M., A. Aroyo, S. Yavin, and Z. Roth. "Seasonal effects on gene expression, cleavage timing, and developmental competence of bovine preimplantation embryos." REPRODUCTION 140, no. 1 (July 2010): 73–82. http://dx.doi.org/10.1530/rep-10-0055.
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We examined the association between season and expression of genes involved in early embryonic development with an emphasis on cleavage rate and timing of the first embryonic cleavage. In Exp. 1, oocytes were aspirated during the cold (Dec–Apr) and hot (May–Nov) seasons. Matured oocytes were chemically activated and culturedin vitro. The developmental peak to the two- and four-cell stages occurred earlier, with a higher proportion of first-cleaved embryos, during the cold season relative to the hot season (P<0.01). In Exp. 2, a time-lapse system was employed to characterize the delayed cleavage noted for the hot season. Cleavage to the two-cell stage occurred in two distinct waves: early cleavage occurred between 18 and 25 h post activation, and late cleavage occurred between 27 and 40 h post activation. In Exp. 3, oocytes were aspirated during the cold and hot seasons, maturedin vitro, fertilized, and cultured for 8 days. In each season, early- and late-cleaved two-cell stage embryos were collected. Total RNA was isolated, and semi-quantitative and real-time PCRs were carried out with primers forGDF9,POU5F1, andGAPDHusing18S rRNAas the reference gene. In both seasons, the expression of all examined genes was higher (P<0.05) in early- versus late-cleaved embryos.POU5F1expression was higher (P<0.05) in early-cleaved embryos developed in the cold season versus the hot season counterparts. The findings suggest a deleterious seasonal effect on oocyte developmental competence with delayed cleavage and variation in gene expression.
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Skubic, Lucijan, Lea Hošnjak, Jeannette P. Staheli, Michael R. Dyen, Rebecca M. Ducore, Lois M. A. Colgin, Anne D. Lewis, and Mario Poljak. "Molecular and Phylogenetic Characterization of Novel Papillomaviruses Isolated from Oral and Anogenital Neoplasms of Japanese Macaques (Macaca fuscata)." Viruses 13, no. 4 (April 7, 2021): 630. http://dx.doi.org/10.3390/v13040630.
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Papillomaviruses (PVs) are a diverse group of host species-specific DNA viruses, etiologically linked with various benign and malignant neoplasms of cutaneous and mucosal epithelia. Here, we describe the detection and characterization of the first two PVs naturally infecting Japanese macaques (Macaca fuscata), including the determination of their etiological association(s) with the development of original neoplasms. The molecular and phylogenetic analyses were performed on complete genome sequences of Macaca fuscata PV types 1 (MfuPV1) and 2 (MfuPV2), which were completely sequenced in samples of a malignant oral tumor and benign anogenital neoplasm of Japanese macaques, respectively. Subsequently, two type-specific quantitative real-time PCRs were developed to estimate viral loads of MfuPV1 and MfuPV2 and to evaluate their etiological roles. The in silico molecular analyses revealed that both viral genomes encode characteristic PV proteins with conserved functional domains and have a non-coding genomic region with regulatory sequences to regulate and complete the viral life cycle. However, additional experimental evidence is needed to finally confirm the presence and biological functionality of the molecular features of both novel PVs. While MfuPV1, together with PVs identified in other macaques, is classified into the Alphapapillomavirus (Alpha-PV) species 12, MfuPV2 is most likely a representative of the novel viral species within the Alpha-PV genus. Their relatively high viral loads suggest that both PVs are etiologically linked with the development of the original neoplasms.
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Wang, Meng. "The effect of Sep15 gene silencing on the expression of crystall oid protein in human lens epithelial cells." E3S Web of Conferences 185 (2020): 03005. http://dx.doi.org/10.1051/e3sconf/202018503005.
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Objective: To investigate the effect of Sep15 gene silencing on lens protein expression in human lens epithelial cells. Method: Human lens epithelial cell (HLEC) SRA01/04 was used as the study object, Using MTT method, RT-PCRs, Small molecule RNA interference technology and protein immunoblotting method, The survival rate of hLE cells stimulated by different concentrations of bFGF and Tm (tunicamycin, an endoplasmic reticulum stress inducing reagent), the changes of the mRNA and protein expression levels of intracellular beta-crystallin and GRP78 proteins, and the changes of Sep15 protein expression levels in hLE cells were determined, respectively, to evaluate the effect of Sep15 gene silencing on the expression of lens protein in human lens epithelial cells. Result: First, The survival rate of hLE cells induced by endoplasmic reticulum stress inducing reagents tunicamycin (Tm) and bFGF was investigated by MTT method. The results showed that a certain concentration of Tm and bFGF could inhibit or lead to the death of hLE cells; Secondly, The effects of Tm on the levels of mRNA and protein expression of the endoplasmic reticulum stress marker protein GRP78 and the effects of bFGF on the levels of alpha-, were detected by real-time fluorescence quantitative PCR and protein immunoblotting, respectively, Effects of beta-crystallin mRNA and protein expression levels, To determine the optimal concentration and time of Tm and bFGF on hLE cells, The results showed that 40 ng/mL of bFGF-treated cells for 48 h was the optimal reaction condition for bFGF to stimulate the differentiation of lens epithelial cells. Third, The co-action of Tm and bFGF on hLE cells was detected by real-time fluorescence quantitative PCR and protein immunoblotting. For Intracellular GRP78, α-, β- crystallin, Effects of mRNA and protein expression levels, The results showed that the occurrence of ER stress could upregulate its expression. Fourth, The changes of Sep15 protein expression level in hLE cells after Sep15 gene silencing and the effects of Tm and bFGF on Sep15 gene silencing in cells were detected by Western blotting. The results showed that the addition of Tm and bFGF basically did not affect the silencing effect of Sep15 gene. Fifth, The expression of GRP78 and β- crystallin in hLE cells after silencing of Sep15 gene was detected by Western blot. The results showed that Sep15 may be involved in the process of protecting the differentiation of lens epithelial cells. Conclusion: Sep15 gene silencing has an inhibitory effect on lens protein expression in human lens epithelial cells, and strengthening its clinical research is of positive significance for improving the clinical treatment and prevention of cataract diseases.
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Ou, Guochun, Qin Liu, Chengxiu Yu, Xiaoju Chen, Wenbo Zhang, Yong Chen, Tao Wang, et al. "The Protective Effects of Maresin 1 in the OVA-Induced Asthma Mouse Model." Mediators of Inflammation 2021 (February 10, 2021): 1–11. http://dx.doi.org/10.1155/2021/4131420.
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Asthma is a chronic inflammatory disease that cannot be cured. Maresin 1 (MaR1) is a specific lipid synthesized by macrophages that exhibits powerful anti-inflammatory effects in various inflammatory diseases. The goal of this study was to evaluate the effect of MaR1 on allergic asthma using an ovalbumin- (OVA-) induced asthma model. Thirty BALB/c mice were randomly allocated to control, OVA, and MaR1 + OVA groups. Mice were sacrificed 24 hours after the end of the last challenge, and serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected for further analysis. Western blotting was used to measure the protein level of IκBα, the activation of the NF-κB signaling pathway, and the expression of NF-κB downstream inflammatory cytokines. Quantitative real-time polymerase chain reactions (qRT-PCRs) were used to evaluate the expression levels of COX-2 and ICAM-1 in lung tissues. We found that high doses of MaR1 were most effective in preventing OVA-induced inflammatory cell infiltration and excessive mucus production in lung tissue, reducing the number of inflammatory cells in the BALF and inhibiting the expression of serum or BALF-associated inflammatory factors. Furthermore, high-dose MaR1 treatment markedly suppressed the activation of the NF-κB signaling pathway, the degradation of IκBα, and the expression of inflammatory genes downstream of NF-κB, such as COX-2 and ICAM-1, in the OVA-induced asthma mouse model. Our findings indicate that MaR1 may play a critical role in OVA-induced asthma and may be therapeutically useful for the management of asthma.
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Pan, Yiou, Xiaochun Zeng, Shuyuan Wen, Xuemei Liu, and Qingli Shang. "Characterization of the Cap ‘n’ Collar Isoform C gene in Spodoptera frugiperda and its Association with Superoxide Dismutase." Insects 11, no. 4 (April 2, 2020): 221. http://dx.doi.org/10.3390/insects11040221.
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Nuclear factor erythroid 2 related factor 2 (Nrf2) belongs to the cap ‘n’ collar basic region leucine zipper (CNC-bZIP) transcription factor family, and is activated by diverse oxidants, pro-oxidants, antioxidants, and chemo-preventive agents. Transcriptional regulation of a battery of detoxifying and antioxidant genes by Nrf2 has been shown to be important for protection against oxidative stress or chemically-induced cellular damages. In our research, we cloned the full length CncC gene from the Spodoptera frugiperda, named as SfCncC. The cDNA of the SfCncC consists of 2652 nucleotides that include a 2196-nucleotide open reading frame (ORF), encoding 731 amino acid residues, and 239- and 217-bp non-coding regions flanking at the 5’- and 3’-ends of the cDNA, respectively. Sequence analysis indicated SfCncC has the conserved domain (CNC-bZIP domain and a tetrapeptide motif, ETGE) character of Nrf2 and showed high identity compared with the CncC/Nrf2 from other insect and vertebrate species. Over-expression of SfCncC can up-regulate the transcription and activity of the SOD gene in Sf9 cells, and the RNAi of SfCncC in Sf9 cells and larvae of S. frugiperda can dramatically reduce the transcriptional level and activity of the SOD gene, as determined by real-time quantitative PCRs. So the SfCncC is involved in the Keap1-Nrf2-ARE pathway, acting the same as the transcriptional factor Nrf2 in vertebrate, and plays a role for host cell defense. The functional characterization of SfCncC provides the fundamental basis for us to further understand the regulatory mechanism of anti-oxidants and anti-xenobiotics in S. frugiperda.
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Andersson, Ann-Catrin, Zhihong Yun, Göran O. Sperber, Erik Larsson, and Jonas Blomberg. "ERV3 and Related Sequences in Humans: Structure and RNA Expression." Journal of Virology 79, no. 14 (July 2005): 9270–84. http://dx.doi.org/10.1128/jvi.79.14.9270-9284.2005.
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ABSTRACT The ERV3 locus at chromosome 7q11 is a much studied human endogenous retroviral (HERV) sequence, owing to an env open reading frame (ORF) and placental RNA and protein expression. An analysis of the human genome demonstrated that ERV3 is one of a group of 41 highly related elements (ERV3-like HERVs) which use proline, isoleucine, or arginine tRNA in their primer binding sites. In addition to elements closely related to ERV3, the group included the previously known retinoic acid-inducible element, RRHERVI, also referred to as HERV15, but was separate from the related HERV-E elements. The ERV3-like elements are defective. The only element with an ORF among gag, pro, pol, and env genes was the env ORF of the original ERV3 locus. A search in dbEST revealed ERV3 RNA expression in placenta, skin, carcinoid tumor, and adrenal glands. Expression was also studied with newly developed real-time quantitative PCRs (QPCR) of ERV3 and HERV-E(4-1) env sequences. Results from a novel histone 3.3 RNA QPCR result served as the expression control. QPCR results for ERV3 were compatible with previously published results, with a stronger expression in adrenal gland and placenta than in 15 other human tissues. The expression of the envelope (env) of ERV3 at chromosome 7q11 was also studied by using stringent in situ hybridization. Expression was found in corpus luteum, testis, adrenal gland, Hassal's bodies in thymus, brown fat, pituitary gland, and epithelium of the lung. We conclude that ERV3 env is most strongly expressed in adrenal and sebaceous glands as well as in placenta.
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Tanabe, Hiroki, Andre J. Ouellette, Melanie J. Cocco, and W. Edward Robinson. "Differential Effects on Human Immunodeficiency Virus Type 1 Replication by α-Defensins with Comparable Bactericidal Activities." Journal of Virology 78, no. 21 (November 1, 2004): 11622–31. http://dx.doi.org/10.1128/jvi.78.21.11622-11631.2004.
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ABSTRACT In addition to their antibacterial activities, certain antimicrobial peptides inactivate enveloped viruses, including the human immunodeficiency virus (HIV). To determine whether peptide bactericidal activities are predictive of antiviral activity, the anti-HIV properties of recombinant human α-defensin 5, mouse α-defensins, cryptdins (Crp) 3 and 4, and rhesus macaque myeloid α-defensins (RMADs) 3 and 4 were determined in vitro. The peptides, purified to homogeneity, had equivalent bactericidal activities that were similar to those of the native molecules. Nuclear magnetic resonance spectroscopy showed RMAD-4 and Crp3 had characteristic α-defensin tridisulfide arrays. Of the peptides analyzed, only RMAD-4 inhibited HIV infectivity at 150 μg/ml, and Crp3 unexpectedly increased HIV replication. Quantitative real-time PCRs for minus-strand strong stop DNA and complete viral cDNA synthesis were used to distinguish between preentry and postentry anti-HIV effects by RMAD-4. Viral exposure to RMAD-4 for 1 h prior to infection reduced HIV minus-strand strong stop DNA and HIV cDNA by 4- to 20-fold during the first round of replication, showing that RMAD-4-exposed virions were not entering cells during the first 24 h. On the other hand, when RMAD-4 was added coincident with HIV inoculation, no anti-HIV activity was detected. Viral exposure to Crp3 resulted in a threefold increase in both HIV minus-strand strong stop DNA and HIV cDNA over the first round of replication. Therefore, two α-defensins, RMAD-4 and Crp3, inhibit or augment HIV replication, respectively, by mechanisms that precede reverse transcription.
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Lee, Jae Il, and In Seop Kim. "TaqMan Probe Real-Time PCR for Quantitative Detection of Mycoplasma during Manufacture of Biologics." KSBB Journal 29, no. 5 (October 30, 2014): 361–71. http://dx.doi.org/10.7841/ksbbj.2014.29.5.361.
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Dong, Xiaojing, Jianqiao Wang, Peng Ji, Longsheng Sun, Shuyan Miao, Yanju Lei, and Xuedi Du. "Seawater Culture Increases Omega-3 Long-Chain Polyunsaturated Fatty Acids (N-3 LC-PUFA) Levels in Japanese Sea Bass (Lateolabrax japonicus), Probably by Upregulating Elovl5." Animals 10, no. 9 (September 17, 2020): 1681. http://dx.doi.org/10.3390/ani10091681.
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The fatty acid compositions of the fish muscle and liver are substantially affected by rearing environment. However, the mechanisms underlying this effect have not been thoroughly described. In this study, we investigated the effects of different culture patterns, i.e., marine cage culture and freshwater pond culture, on long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis in an aquaculturally important fish, the Japanese sea bass (Lateolabrax japonicus). Fish were obtained from two commercial farms in the Guangdong province, one of which raises Japanese sea bass in freshwater, while the other cultures sea bass in marine cages. Fish were fed the same commercial diet. We found that omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) levels in the livers and muscles of the marine cage cultured fish were significantly higher than those in the livers and muscles of the freshwater pond cultured fish. Quantitative real-time PCRs indicated that fatty acid desaturase 2 (FADS2) transcript abundance was significantly lower in the livers of the marine cage reared fish as compared to the freshwater pond reared fish, but that fatty acid elongase 5 (Elovl5) transcript abundance was significantly higher. Consistent with this, two of the 28 CpG loci in the FADS2 promoter region were heavily methylated in the marine cage cultured fish, but were only slightly methylated in freshwater pond cultured fish (n = 5 per group). Although the Elovl5 promoter was less methylated in the marine cage reared fish as compared to the freshwater pond reared fish, this difference was not significant. Thus, our results might indicate that Elovl5, not FADS2, plays an important role in the enhancing LC-PUFA synthesis in marine cage cultures.
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Galtseva, I. V., M. L. Filipenko, Yu O. Davydova, A. V. Luchkin, N. M. Kapranov, Yu A. Kondratieva, S. V. Subbotin, et al. "Comparison of polymerase chain reaction and flow cytometry for measuring telomere length of human leukocytes." Russian Clinical Laboratory Diagnostics 66, no. 3 (March 30, 2021): 154–59. http://dx.doi.org/10.51620/0869-2084-2021-66-3-154-159.
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Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.
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Choi, Yan Ru, Min-Chun Chen, Maura Carrai, Francesca Rizzo, Yingfei Chai, May Tse, Ken Jackson, et al. "Hepadnavirus DNA Is Detected in Canine Blood Samples in Hong Kong but Not in Liver Biopsies of Chronic Hepatitis or Hepatocellular Carcinoma." Viruses 14, no. 7 (July 15, 2022): 1543. http://dx.doi.org/10.3390/v14071543.
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Chronic hepatitis and hepatocellular carcinoma (HCC) caused by the hepadnavirus hepatitis B virus (HBV) are significant causes of human mortality. A hepatitis-B-like virus infecting cats, domestic cat hepadnavirus (DCH), was reported in 2018. DCH DNA is hepatotropic and detectable in feline blood or serum (3.2 to 12.3%). Detection of HBV DNA has been reported in sera from 10% of free-roaming dogs in Brazil, whereas 6.3% of sera from dogs in Italy tested positive for DCH DNA by real-time quantitative PCR (qPCR). If DCH, HBV, or another hepadnavirus is hepatotropic in dogs, a role for such a virus in the etiology of canine idiopathic chronic hepatitis (CH) or HCC warrants investigation. This study investigated whether DCH DNA could be detected via qPCR in blood from dogs in Hong Kong and also whether liver biopsies from dogs with confirmed idiopathic CH or HCC contained hepadnaviral DNA using two panhepadnavirus conventional PCRs (cPCR) and a DCH-specific cPCR. DCH DNA was amplified from 2 of 501 (0.4%) canine whole-blood DNA samples. A second sample taken 6 or 7 months later from each dog tested negative in DCH qPCR. DNA extracted from 101 liver biopsies from dogs in Hong Kong or the USA, diagnosed by board-certified pathologists as idiopathic CH (n = 47) or HCC (n = 54), tested negative for DCH DNA and also tested negative using panhepadnavirus cPCRs. This study confirms that DCH DNA can be detected in canine blood by qPCR, although at a much lower prevalence than that reported previously. We identified no evidence to support a pathogenic role for a hepadnavirus in canine idiopathic CH or HCC.
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Boettcher, Sebastian, Sebastian Irmer, Silke Lueschen, Matthias Ritgen, Thorsten Raff, Nicola Goekbuget, Dieter Hoelzer, Heinz-August Horst, Michael Kneba, and Monika Brueggemann. "Sensitivity and Applicability of Six-Color Flow Cytometry Is Comparable to ASO-Primer-Real-Time PCR (RQ-PCR) for Minimal Residual Disease (MRD) Monitoring in Adult Acute Lymphoblastic Leukemia (ALL) - A Comparative Analysis in 70 Patients from the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL)." Blood 108, no. 11 (November 1, 2006): 2287. http://dx.doi.org/10.1182/blood.v108.11.2287.2287.
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Abstract Although the quantification of MRD by PCR or flow cytometry (FC) is of proven prognostic value in ALL, both methods were never compared in adults. Since 3- to 4-color FC showed equal or inferior sensitivity to PCR in childhood ALL, we herein prospectively compared six-color FC (6C-FC) at initial diagnosis and during early follow-up in adult ALL to RQ-PCR as reference method. Diagnostic samples from 70 consecutive ALL patients (pts) (22 T-, 48 B-lineage; 18 to 71 yrs) were screened by 14 different multiplex immungene consensus PCRs as published (Bruggemann et al., Blood, 2006) and simultaneously by 8 different 6C-FC combinations which included a total of 29 different antibodies. At least one of the consensus PCRs revealed monoclonality in 64/70 pts (91 %), whereas immunophenotypic aberrations could distinguish ALL from benign hematopoiesis in 67/70 cases (96 %). 6C-FC was positive in all PCR negative samples, while PCR was successful in all samples lacking immunophenotypic aberrations. The standardized 6C-FC method revealed a median of 4 immunophenotypic aberrations per pt (range 1 to 8). The most frequently observed aberrations in B-lineage ALL included CD58++ (76 %), CD11a++(50%), CD22++ (44 %), low CD38 (50%), and KORSA+ (35 %). Tdt+cytoplasmatic(cy)CD3+ hallmarked 86% of T-lineage ALL cases which were additionally characterized by CD7+ (100%), surface(s) CD3- (90%), CD99+ (52 %), and CD1a+ (38 %). Up to now, RQ-PCR assays have been established for 38 pts (median sensitivity 10−4; range 10−5 to 10−2) thus allowing for comparisons of MRD assessments to 6C-FC in 155 follow-up samples. 110/155 (71 %) samples were collected during the first 4 therapy months. 6C-FC marker combinations for follow-up always included CD34/CD10/CD19/CD45 in B-lineage and TdT/cyCD3/sCD3/CD5 in T-lineage ALL. Stainings were individualized by adding 2 pt-specific markers each to up to 3 tubes. A total of 155 follow-up specimens comprised 13 FC-/RQ-PCR+ (8 %), 4 FC+/RQ-PCR- (3 %), 88 concordantly negative (57 %), and 50 concordantly positive (32 %) samples. Due to extremely low MRD, in 11/13 FC-/RQ-PCR+ samples (85 %) the results of RQ-PCR were qualitatively positive only, but not accurately quantifiable. The minimum MRD level detectable by 6C-FC was 3x10−5, as proven by a concordantly positive RQ-PCR result. MRD levels obtained by both methods correlated well (Spearman r = 0.85; p < 0.0001) in follow-up samples that were positive both by RQ-PCR and by 6C-FC. The median ratio between 6C-FC and RQ-PCR MRD results was 0.48 (range 0.012 to 113). In 11% of samples the ratio between MRD levels obtained by the two methods differed more than tenfold. Our novel standardized 6C-FC approach is highly sensitive for MRD assessments in adult ALL. Our results suggest the applicability of 6C-FC in a multicenter setting, an excellent specificity, a good quantitative correlation and a similar sensitivity when compared to RQ-PCR. Longer follow-up and the inclusion of more samples in particular from later time points are required to decide whether or not the two methods are of comparable clinical significance.
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Kosinova, E., I. Psikal, B. Robesova, and K. Kovarcik. "Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry." Veterinární Medicína 52, No. 6 (January 7, 2008): 253–61. http://dx.doi.org/10.17221/1882-vetmed.
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Quantitative real-time RT-PCR (qRT-PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus (BVDV) in clinical samples from persistently infected cattle. qRT-PCR was optimized to quantify the number of BVD virus copies using Light Cycler<sup>®</sup> detection system and intercalation fluorogenic dye SYBR Green I. A universal set of primers was selected from a highly conserved 5′ untranslated region (5′UTR) to detect BVDV type I and II simultaneously. Quantification of BVDV cDNA was accomplished using a calibration curve generated from 10-fold serial dilutions of standard plasmid DNA in the range 1−10<sup>8</sup> copies/μl. Analysis of 290 bp amplicons enabled monitoring of the viral RNA/BVDV level in a total of five BVDV strains (BVD-NADL, A03/3004, DB03/2943, KA04/3124, KV05/3412) and sixteen bulk milk samples, and in bovine sera of persistent carriers originating from Czech farms, as well as in a batch of calf serum for cell culture. Melting temperatures of amplicons (Tm) of BVDV strains of the same genotype group I as the NADL reference strain showed variability of the thermal points, however significant differences were observed in Tm values between the representatives of genotype group I and II. Low concentrations of BVD virus in bulk milk samples were also qualitatively identified by conventional RT-PCR. Highly reproducible data were obtained as the coefficients of variation of threshold cycles values in intra-assay and inter-assay were less than 0.85% and 2.76%, respectively. The results give enough evidence of suitability of qRT-PCR assay for quantitative analysis of BVDV in clinical samples.
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Hung, Pi-Lien, Mei-Hsin Hsu, Hong-Ren Yu, Kay L. H. Wu, and Feng-Sheng Wang. "Thyroxin Protects White Matter from Hypoxic-Ischemic Insult in the Immature Sprague–Dawley Rat Brain by Regulating Periventricular White Matter and Cortex BDNF and CREB Pathways." International Journal of Molecular Sciences 19, no. 9 (August 29, 2018): 2573. http://dx.doi.org/10.3390/ijms19092573.
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Background: Periventricular white-matter (WM) injury is a prominent feature of brain injury in preterm infants. Thyroxin (T4) treatment reduces the severity of hypoxic-ischemic (HI)-mediated WM injury in the immature brain. This study aimed to delineate molecular events underlying T4 protection following periventricular WM injury in HI rats. Methods: Right common-carotid-artery ligation, followed by hypoxia, was performed on seven-day-old rat pups. The HI pups were injected with saline, or 0.2 or 1 mg/kg of T4 at 48–96 h postoperatively. Cortex and periventricular WM were dissected for real-time (RT)-quantitative polymerase chain reactions (PCRs), immunoblotting, and for immunofluorescence analysis of neurotrophins, myelin, oligodendrocyte precursors, and neointimal. Results: T4 significantly mitigated hypomyelination and oligodendrocyte death in HI pups, whereas angiogenesis of periventricular WM, observed using antiendothelium cell antibody (RECA-1) immunofluorescence and vascular endothelium growth factor (VEGF) immunoblotting, was not affected. T4 also increased the brain-derived neurotrophic factors (BDNFs), but not the nerve growth factor (NGF) expression of injured periventricular WM. However, phosphorylated extracellular signal regulated kinase (p-ERK) and phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) concentrations, but not the BDNF downstream pathway kinases, p38, c-Jun amino-terminal kinase (c-JNK), or Akt, were reduced in periventricular WM with T4 treatment. Notably, T4 administration significantly increased BDNF and phosphorylated CREB in the overlying cortex of the HI-induced injured cortex. Conclusion: Our findings reveal that T4 reversed BNDF signaling to attenuate HI-induced WM injury by activating ERK and CREB pathways in the cortex, but not directly in periventricular WM. This study offers molecular insight into the neuroprotective actions of T4 in HI-mediated WM injury in the immature brain.
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Timar, Botond, Amy Chadburn, Daniel Knowles, and Ethel Cesarman. "Activation of Classical and Alternative Nuclear Factor-kappaB (NF-kB) Pathways in Diffuse Large B-Cell Lymphomas." Blood 104, no. 11 (November 16, 2004): 29. http://dx.doi.org/10.1182/blood.v104.11.29.29.
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Abstract Activation of the NF-kB pathway is involved in many human neoplasms. In this study we examined the status of the NF-kB canonical (IkB, p50/p65) and non-canonical (p52, RelB) pathways in diffuse large B-cell lymphomas (DLBCL), which are a common and heterogeneous group of lymphoid malignancies. DLBCL have been divided into activated B-cell (ABC) like, and germinal center B-cell (GCB) like subgroups, which have been reported to have high and low NF-kB activity, respectively. However, the nature of the NF-kB complexes in this lymphoma entity has not been previously evaluated. Therefore we performed Western blotting, electrophoretic mobility shift assays (EMSA) and real time quantitative RT-PCRs on nuclear and cytoplasmic protein and total RNA extracts in 20 primary tumor samples and 9 different DLBCL cell lines. In the cell lines, presence of NF-kB proteins in nuclear extracts correlated with expression of NF-kB target genes (CCR7, IkBa, CCND2, BCL-2, IRF4) as determined by RT-PCR. Therefore, these could be assigned into GCB and ABC-like categories. In primary DLBCLs, EMSA showed NF-kB binding in all but one case. The same cases (19/20) had high p52 in the nucleus indicating activation of the alternative pathway. TNF family ligand BAFF was also found to be expressed in all primary samples and most cell lines. The classical pathway, as determined by nuclear p50, was also present in these cases. Levels of p65 and RelB expression were variable, but did not correlate with the mRNA expression of NF-kB target genes. In conclusion, while DLBCL cell lines may be divided into two distinct categories, the primary samples represented a spectrum of NF-kB target gene activity, while levels of NF-kB expression were high. BAFF expression and activation of the alternative pathway may be important in the pathogenesis of DLBCL.
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Kleeberger, J. A., J. Neuser, D. de Gonzalo-Calvo, T. Kempf, J. Bauersachs, T. Thum, and J. D. Widder. "microRNA-206 correlates with left ventricular function after transcatheter aortic valve implantation." American Journal of Physiology-Heart and Circulatory Physiology 313, no. 6 (December 1, 2017): H1261—H1266. http://dx.doi.org/10.1152/ajpheart.00432.2017.
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Transcatheter aortic valve implantation (TAVI) is the method of choice in patients with high risk or contraindications for conventional aortic valve replacement. However, it is not well understood which parameters predict the overall cardiac function postprocedurally. miRNAs are small noncoding RNA molecules that repress gene expression by different mechanisms and can also be detected in the blood. Recent studies have shown that miRNAs detected in the blood may serve as sensitive and specific biomarkers in various diseases; therefore, we examined the levels of different microRNAs in the serum of patients undergoing TAVI. We thereby intended to find potential predictors for cardiac function after TAVI. Serum from patients with aortic valve disease was obtained at five different points: before the TAVI procedure, at days 1 and 3 after the TAVI procedure, and the day of dischargement and after a period of 3 mo. We next performed quantitative real-time PCRs to examine the samples for changes in the level of miRNAs previously described as cardiac enriched. Our results show that the level of miR-206 in the serum of patients after TAVI correlated negatively with the left ventricular ejection fraction of individual patients. We found left ventricular function to be better in patients with lower levels of miR-206 after implantation of the new valve. A decrease in the serum level of miR-206 may be linked to changes in cardiac function of patients after TAVI. Further studies are necessary to test the miRNA for its potential value as a prognostic marker. NEW & NOTEWORTHY This study is the first to investigate novel miRNA-based biomarkers within the context of transcatheter aortic valve implantation. miRNA-206 proved to correlate inversely with the postprocedural left ventricular ejection fraction of patients.